Method for preparing cells with high expression of nnmt, nnmt-rich exosomes and applications thereof

Abstract

The application provides a cell with high expression of NNMT, a preparation method of exosomes rich in NNMT and application of the exosomes. It is found for the first time that Taizishen cyclic peptide B can promote expression of NNMT, based on which, the application provides a preparation method of a cell with high expression of NNMT and a preparation method of exosomes rich in NNMT for the field, and it is verified through experiments that the exosomes rich in NNMT can effectively treat vascular aging or a disease or a symptom related to vascular aging.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the preparation method of cells that highly express NNMT, NNMT-rich exosomes, and their applications. Background Technology

[0002] Blood vessels are not only conduits for transporting blood, but also multifunctional organs. The relative balance of vascular homeostasis is a crucial foundation for maintaining normal physiological functions. Imbalance in vascular homeostasis not only affects a single organ but can also cause systemic panvascular diseases. Vascular aging refers to a series of degenerative changes in the morphology and function of blood vessels with age, mainly including thickening of the vessel walls, decreased elasticity, increased stiffness, and endothelial dysfunction. Vascular aging increases the risk of developing various diseases.

[0003] Some common diseases related to vascular aging include atherosclerosis, hypertension, atrial fibrillation and heart failure, chronic kidney disease, cerebrovascular disease, cognitive decline, neurological diseases such as Alzheimer's disease, and peripheral vascular disease. Therefore, the development of intervention strategies targeting vascular aging is of great significance for the prevention and treatment of various major diseases. Summary of the Invention

[0004] In view of this, in order to overcome the shortcomings of the prior art, the present invention is proposed.

[0005] The first aspect of the present invention provides a method for preparing cells that highly express nicotinamide-N-methyltransferase (NNMT), the method comprising mixing Heterophyllin B (HPB) and / or Codonopsis pilosula extract, and cells.

[0006] In some implementations, the concentration of the ginseng cyclic peptide B can be any concentration that can promote NNMT expression.

[0007] In some embodiments, the concentration of the Codonopsis pilosula cyclic peptide B used is 1 nM-100 μM.

[0008] In some embodiments, the concentration of the ginseng cyclic peptide B is selected from 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 20 μM, 50 μM, and 100 μM.

[0009] In some embodiments, the Codonopsis pilosula extract contains Codonopsis pilosula cyclic peptide B.

[0010] In some implementations, the extract of Codonopsis pilosula is obtained by means of raw material pretreatment, solvent extraction, preliminary concentration, separation and purification (enrichment of cyclic peptide B), and obtaining the extract.

[0011] In some embodiments, the effective concentration of ginseng cyclic peptide B in the ginseng extract is 1 nM-100 μM.

[0012] In some embodiments, the effective concentration of ginseng cyclic peptide B in the ginseng extract is selected from 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 20 μM, 50 μM, and 100 μM.

[0013] In some implementations, the mixing time can be any time that promotes NNMT expression.

[0014] In some implementations, the mixing time is 0.5-96 hours.

[0015] In some implementations, the mixing time is 24-48 hours.

[0016] In some implementations, the method further includes passage culture of the cells.

[0017] In some implementations, after passage culture, when the cell confluence reaches 80%-90%, Codonopsis pilosula cyclic peptide B and / or Codonopsis pilosula extract are mixed with the cells.

[0018] In some implementations, the cells include vascular endothelial cells.

[0019] In some embodiments, the vascular endothelial cells include, but are not limited to, HUAECs, HCAECs, HAAECs, HDMECs, HBMECs, HRMECs, HPMECs, and HIMECs.

[0020] In some embodiments, the cells are selected from HUVECs cells.

[0021] A second aspect of the present invention provides a method for preparing NNMT-rich exosomes, the method comprising collecting exosomes generated from cells prepared by the method described in the first aspect of the present invention.

[0022] In some implementations, the collection methods include, but are not limited to, ultracentrifugation, density gradient centrifugation, size exclusion chromatography, polymer precipitation, immunomagnetic bead method, and ultrafiltration.

[0023] In some implementations, the above method steps are performed in accordance with steps commonly used by those skilled in the art.

[0024] In some implementations, the principle of ultracentrifugation is to gradually remove cells, cell debris, and larger apoptotic bodies by using centrifugal forces at different speeds, and finally precipitate exosomes at ultra-high speeds (typically ≥100,000 g) using specific centrifugal forces. This includes steps such as low-speed centrifugation (cell removal), medium-speed centrifugation (debris removal), and high-speed ultracentrifugation (exosome collection).

[0025] In some embodiments, the principle of density gradient centrifugation is to use a medium (such as sucrose or iodixanol) to form a density gradient before or during ultracentrifugation. Because exosomes have a unique buoyant density (1.10-1.21 g / mL), they float to a gradient layer of equal density after ultracentrifugation, thus separating from other impurities.

[0026] In some implementations, the principle of size exclusion chromatography is to use a chromatographic column packed with porous gel to separate particles based on their size. Large particles cannot enter the gel pores and are eluted first; small particles enter the pores, have a longer path, and are eluted later. Exosomes are of uniform size and are collected in a specific eluent.

[0027] In some embodiments, the polymer precipitation method includes polyethylene glycol (PEG) precipitation. The principle of PEG precipitation is to use a hydrophilic polymer "network" such as polyethylene glycol to encapsulate the exosomes, reduce their solubility, and then precipitate them by low-speed centrifugation.

[0028] In some implementations, the principle of immunomagnetic bead assay is to use magnetic beads coated with specific antibodies to bind to antigens on the surface of exosomes and separate them under the action of an external magnetic field.

[0029] In some implementations, the principle of ultrafiltration is to use an ultrafiltration membrane with a specific pore size (typically 10-100 kDa or 200-500 nm) to allow small molecules (proteins, salts) to pass through the membrane by centrifugal force or pump pressure, while large particles such as exosomes are retained and concentrated.

[0030] In some implementations, the collection method is selected from ultracentrifugation and size exclusion chromatography.

[0031] A third aspect of this invention provides a drug screening method for detecting drugs that enhance the expression of NNMT by codonopsis pilosula cyclic peptide B, the drug screening method comprising: Mixed cells that had been treated with Codonopsis pilosula cyclic peptide B with test compounds; Detect the expression level of NNMT in cells; The detected expression levels were compared with the NNMT expression levels obtained in the absence of the test compound to determine whether the test compound was a drug that promoted the enhancement of NNMT expression by ginseng cyclic peptide B or a drug that inhibited the enhancement of NNMT expression by ginseng cyclic peptide B.

[0032] In some embodiments, the determination method for the test compound being a drug that enhances the expression of NNMT by codonopsis pilosula cyclic peptide B is that the NNMT expression level detected after the addition of the test compound is higher than the NNMT expression level when the test compound is not present.

[0033] In some embodiments, the method for determining whether the test compound is a drug that inhibits the enhancing effect of codonopsis cyclopeptide B on NNMT expression is that the NNMT expression level detected after adding the test compound is lower than the NNMT expression level when the test compound is not present.

[0034] In some embodiments, the compound includes small molecule compounds and macromolecule compounds.

[0035] In some embodiments, the macromolecular compound includes proteins, DNA, and RNA.

[0036] In some embodiments, the protein includes synthetically produced protein analogs or naturally occurring proteins. The protein analogs have similar functions or activities to the proteins.

[0037] In some implementations, the protein includes, but is not limited to, antibodies.

[0038] In some implementations, the DNA includes single-stranded DNA, closed circular DNA, and linker DNA.

[0039] In some implementations, the RNA includes mRNA, tRNA, rRNA, snRNA, hRNA, antisense RNA, tCRNA, dsRNA, SCRNA, catalytically active RNA, and various viral RNAs.

[0040] In some implementations, the antibody includes, but is not limited to, dAb, Fab, Fab', scFv, Fv, disulfide-bonded Fv, or containing a single immunoglobulin variable domain.

[0041] The fourth aspect of the present invention provides any of the following methods: (1) A method for regulating NNMT expression in in vitro cells, the method comprising mixing cells and Codonopsis pilosula cyclic peptide B; (2) A drug screening method, wherein the screening method is based on NNMT, and the drug is a drug for treating vascular aging or vascular aging-related diseases or conditions, specifically including: Obtain NNMT initiation expression level data in cells 1; Mixing cells and test compounds; The expression level of NNMT in cells was detected, and expression level data were obtained. Compare expression level data 2 with expression level data 1. If expression level data 2 is higher than expression level data 1, then the test compound is a drug for treating vascular aging or vascular aging-related diseases or conditions. In some implementations, the above method also includes culturing the mixed cells; (3) A method for predicting the sensitivity of vascular aging or vascular aging-related diseases or conditions to the drug Pseudostellaria heterophylla cyclopeptide B, the method comprising detecting the expression level of NNMT after administration of Pseudostellaria heterophylla cyclopeptide B.

[0042] In some implementations, the cell can be any cell capable of expressing NNMT. This expression can be either a cell that does not express NNMT itself, but expresses NNMT after being influenced by other substances such as the test compound or Codonopsis pilosula cyclic peptide B, or a cell that expresses NNMT itself, but whose expression level is altered after being influenced by other substances such as the test compound or Codonopsis pilosula cyclic peptide B.

[0043] In some implementations, the cells include vascular endothelial cells.

[0044] In some embodiments, the vascular endothelial cells include, but are not limited to, HUAECs, HCAECs, HAAECs, HDMECs, HBMECs, HRMECs, HPMECs, and HIMECs.

[0045] In some embodiments, the cells are selected from HUVECs cells.

[0046] In some implementations, the vascular aging can be physiological or pathological. Physiological vascular aging is caused by programmed aging. Pathological vascular aging is caused by environmental factors, nutrition, and disease, which accelerate the aging of blood vessels, leading to premature aging and pathological changes in their nature.

[0047] In some implementations, the vascular aging can lead to vascular aging-related diseases or conditions.

[0048] In some implementations, vascular aging can lead to, but is not limited to, atherosclerosis, hypertension, stroke, tumors, lower limb ischemia, and diabetic nephropathy.

[0049] In some implementations, the vascular aging-related diseases or conditions are selected from lower limb ischemia, diabetic nephropathy, etc.

[0050] The fifth aspect of the present invention provides any of the following products: (1) A system and apparatus for producing cells that highly express NNMT, the system and apparatus comprising: Mixed unit: Mixed cells with Codonopsis pilosula cyclic peptide B; Culture unit: culturing the mixed cells; (2) A system or apparatus for producing exosomes rich in NNMT, the system or apparatus comprising: Cell acquisition unit: Acquires cells prepared by the preparation method described in the first aspect of the present invention; Collection unit: collects exosomes produced by cells prepared by the preparation method described in the first aspect of the present invention; (3) A drug screening system for screening drugs that enhance the expression of NNMT by Codonopsis pilosula cyclic peptide B, the system comprising: Mixing unit: Mixing cells treated with Codonopsis pilosula cyclic peptide B with the test compound; Detection unit: Detects the expression level of NNMT in cells; Drug screening unit: The detected expression level results are compared with the NNMT expression level obtained in the absence of the test compound to identify the test compound as either a drug that promotes the enhancement of NNMT expression by ginseng cyclic peptide B or a drug that inhibits the enhancement of NNMT expression by ginseng cyclic peptide B. In some embodiments, the method for determining whether the test compound is a drug that enhances the expression of NNMT by codonopsis pilosula cyclic peptide B is that the NNMT expression level detected after adding the test compound is higher than the NNMT expression level when the test compound is not present. In some embodiments, the method for determining whether the test compound is a drug that inhibits the enhancing effect of codonopsis cyclopeptide B on NNMT expression is that the NNMT expression level detected after adding the test compound is lower than the NNMT expression level when the test compound is not present. (4) A drug screening system, wherein the drug is a drug for treating vascular aging or vascular aging-related diseases or conditions, specifically including: Data acquisition unit: Acquire NNMT initial expression level data of cells 1; Mixing unit: mixing cells with test compounds; Detection unit: Detects the expression level of NNMT in cells and obtains expression level data 2; Drug screening unit: compare expression level data 2 with expression level data 1. If expression level data 2 is higher than expression level data 1, then the test compound is a drug for treating vascular aging or vascular aging-related diseases or conditions. (5) A system or apparatus for predicting the sensitivity of vascular aging or vascular aging-related diseases or conditions to the drug Codonopsis pilosula cyclic peptide B, wherein the system or apparatus specifically includes: Acquisition Unit: Acquire NNMT initial expression level data of cells 1; Drug administration unit: administration of Codonopsis pilosula cyclic peptide B; Detection unit: Detect the expression level of NNMT after administration of Codonopsis pilosula cyclic peptide B, and obtain expression level data 2; Prediction Unit: Compares expression level data 2 with expression level data 1. If expression level data 2 is higher than expression level data 1, it is predicted that the vascular aging or vascular aging-related diseases or conditions are highly sensitive to Codonopsis pilosula cyclic peptide B drug. If expression level data 2 is not higher than expression level data 1, it is predicted that the vascular aging or vascular aging-related diseases or conditions are less sensitive to Codonopsis pilosula cyclic peptide B drug.

[0051] In some embodiments, the compound includes small molecule compounds and macromolecule compounds.

[0052] In some embodiments, the macromolecular compound includes proteins, DNA, and RNA.

[0053] In some embodiments, the protein includes synthetically produced protein analogs or naturally occurring proteins. The protein analogs have similar functions or activities to the proteins.

[0054] In some implementations, the protein includes, but is not limited to, antibodies.

[0055] In some implementations, the DNA includes single-stranded DNA, closed circular DNA, and linker DNA.

[0056] In some implementations, the RNA includes mRNA, tRNA, rRNA, snRNA, hRNA, antisense RNA, tCRNA, dsRNA, SCRNA, catalytically active RNA, and various viral RNAs.

[0057] In some implementations, the antibody includes, but is not limited to, dAb, Fab, Fab', scFv, Fv, disulfide-bonded Fv, or containing a single immunoglobulin variable domain.

[0058] In some implementations, the cells include vascular endothelial cells.

[0059] In some embodiments, the vascular endothelial cells include, but are not limited to, HUAECs, HCAECs, HAAECs, HDMECs, HBMECs, HRMECs, HPMECs, and HIMECs.

[0060] In some embodiments, the cells are selected from HUVECs cells.

[0061] A sixth aspect of the present invention provides a computer device comprising: a memory and a processor, wherein the memory is used to store program instructions; and the processor is used to invoke the program instructions, wherein when the program instructions are executed, the preparation method described in the first aspect of the present invention, the preparation method described in the second aspect of the present invention, the drug screening method described in the third aspect of the present invention, the drug screening method described in the fourth aspect of the present invention, or the prediction method described in the fourth aspect of the present invention.

[0062] A seventh aspect of the present invention provides a computer-readable storage medium having a computer program thereon, comprising: when the computer program is executed by a processor, implementing the preparation method of the first aspect of the present invention, the preparation method of the second aspect of the present invention, the drug screening method of the third aspect of the present invention, the drug screening method of the fourth aspect of the present invention, or the prediction method of the fourth aspect of the present invention.

[0063] The eighth aspect of the present invention provides any of the following products: (1) A cell that highly expresses NNMT, said cell being prepared by the preparation method described in the first aspect of the present invention; (2) An exosome rich in NNMT, said exosome being prepared by the preparation method described in the second aspect of the present invention; (3) A drug obtained based on the drug screening method described in the third aspect of the present invention or the drug screening method described in the fourth aspect of the present invention; (4) A pharmaceutical composition comprising cells that highly express NNMT as described in the eighth aspect (1) of the present invention, NNMT-rich exosomes as described in the eighth aspect (2) of the present invention, or a drug as described in the eighth aspect (3) of the present invention.

[0064] In some embodiments, the pharmaceutical composition further includes a pharmaceutically acceptable carrier and / or excipient.

[0065] Pharmaceutically acceptable carriers and / or excipients include, but are not limited to, preservatives, stabilizers, wetting agents or emulsification promoters, and adjuvants (such as salts and / or buffers used to control osmotic pressure).

[0066] According to conventional methods, this pharmaceutical composition can be formulated into various forms of oral or parenteral administration agents.

[0067] Examples of oral drug delivery systems include, but are not limited to, tablets, pills, hard capsules, soft capsules, liquids, suspensions, emulsions, syrups, powders, microparticles, granules, and pellets. In addition to the active ingredient, these dosage forms may also contain surfactants, diluents (e.g., lactose, dextran, sucrose, mannitol, sorbitol, cellulose, glycine, etc.), lubricants (e.g., silica, talc, stearic acid and its magnesium or calcium salts, and polyethylene glycol). Tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth gum, methylcellulose, sodium carboxymethyl cellulose, and polyvinylpyrrolidone. Tablets may optionally contain pharmaceutical additives such as starch, agar, disintegrants (e.g., alginate or its sodium salt), absorbents, colorants, flavoring agents, and sweeteners. Tablets can be prepared by conventional mixing, granulation, or coating methods.

[0068] In addition, the dosage form for parenteral administration can be a transdermal dosage form, such as, for example, injections, drops, ointments, lotions, gels, creams, sprays, suspensions, oils, suppositories, or patches, but is not limited to these.

[0069] In some implementations, the exosomes also include other therapeutically useful substances.

[0070] In some implementations, the other therapeutically useful substances include, but are not limited to, other drugs for treating atherosclerosis, hypertension, stroke, tumors, lower limb ischemia, or diabetic nephropathy.

[0071] In some implementations, the drugs for treating atherosclerosis include, but are not limited to, statins such as atorvastatin and rosuvastatin, antiplatelet drugs such as aspirin, clopidogrel, and ticagrelor, thrombolytic drugs such as urokinase, streptokinase, and recombinant tissue plasminogen activator, anticoagulants such as heparin, low molecular weight heparin, warfarin, dabigatran etexilate, and rivaroxaban, nitrates, long-acting calcium channel blockers, prostaglandins, and 5-HT2 receptor selective antagonists such as sarpogrelate.

[0072] In some embodiments, the drugs for treating hypertension include, but are not limited to, diuretics such as hydrochlorothiazide and indapamide, calcium channel blockers such as amlodipine and nifedipine, angiotensin-converting enzyme inhibitors such as enalapril and perindopril, angiotensin II receptor antagonists such as valsartan and irbesartan, beta-blockers such as metoprolol and bisoprolol, and alpha-blockers such as terazosin and doxazosin.

[0073] In some embodiments, the medication for treating stroke includes, but is not limited to, medications for treating ischemic stroke.

[0074] In some implementations, the drugs for treating ischemic stroke include, but are not limited to, antiplatelet drugs such as aspirin, clopidogrel, and ticagrelor; statins such as atorvastatin and rosuvastatin; neuroprotective drugs such as edaravone and citicoline; drugs that improve cerebral circulation such as butylphthalide and human urinary kininogenase; and anticoagulants such as heparin and warfarin.

[0075] In some embodiments, the medicaments for treating tumors include, but are not limited to, alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, indomethacin, and piperazine; aziridines such as benzodopa, carbaquinone, metopepa, and uredopa; ethyleneimine and methylmelamine, including hexamethylmelamine, triethylene melamine, triethylphosphamide, triethylthiophosphamide, and trimethylol melamine; anechoic acid lactones (especially anocaryopicrin and bratasinone); camptothecin (including the synthetic analogue topotecan); lichenin; callystatin; CC-1065 (including its synthetic analogues adolaxine, calcetin, and bizelinine); cryptophytic acid (especially macrolide 1 and macrolide 8); dolalastatin; docalamicin (including the synthetic analogue KW-2189). And CB1-TM1); eleutherobin; sarcodictyin; spongistatin; nitrogen mustard, such as chlorambucil, naphthalenemustine, chophosphatamide, estradiol, ifosfamide, nitrogen mustard, oxynitric acid mustard hydrochloride, melphalan, neo-enpichin, cholesterol phenylacetic acid mustard, prednimustine, trofosfamide, and uracil mustard; nitrosourea, such as Carmustine, chloramphenicol, formustine, lomustine, nimustine, and ranola mustard; antibiotics, such as enematic antibiotics (e.g., calcipomycin, especially calcipomycin γI and calcipomycin ωI1); danendomycin, including danendomycin A; bisphosphonates, such as clophosphonates; esporomycin; and new carcinogen chromophores and related chromogens, enematic antibiotic chromophores, aclacinomysins, actinomycins, azoserine, bleomycin, and actinomycin. Carabicin C, erythromycin, carcinomycin, chromomycinis, daunorubicin, detoxin, 6-diazo-5-oxo-L-leucine, doxorubicin (including morpholino-doxorubicin, cyanomolino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, isorubicin, idarubicin, mesorcinol, mitomycin such as mitomycin C, mycophenolic acid, nogamycin, olivomycin, pepromycin, pofibromycin, purine Doxorubicin, triamcinolone acetonide, rodobisacin, streptomycin, strepzotocin, tuberculin, ubenmexicoside, fentostatin, and fentostatin; anti-metabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs, such as folate, pteroxate, and trimethyltroxa; purine analogs, such as fludarabine, 6-mercaptopurine, thioimidazoline, and thioguanine; pyrimidine analogs, such as azacitidine, azacitidine, 6-azouridine, carmoflurane, cytarabine, dideoxyuridine, deoxyfluorouridine, enoxabin, and fluorouridine;Androgens, such as capetestosterone, drotalbutone propionate, cyclothiosterol, meandrazan, and testosterone lactone; anti-adrenergics, such as mitotane and trilosterone; folic acid supplements, such as folinic acid; acetoglucuronolactone; aldehydephosphoramide glycoside; aminolevulinic acid; enuracil; acridine; bestrabucil; dianthraxetine; edaraxazole; desphosphonamide; desmecoxine; desaccharin; epoxetine; edaraxetine; ethoxyphenidate Gallium nitrate; Hydroxyurea; Lentinan; Lolitainine; Maytansine, such as Maytansine and Ansorcinol; Mitoguanidine hydrazone; Mitoanthraquinone; Mopidanmol; Nitraerine; Pensistatin; Methionine mustard; Pirarubicin; Loxoanthraquinone; Podophyllotoxin; 2-Ethylhydrazide; Methylbenzylhydrazide; PSK polysaccharide complex; Razosen; Rhizobium; Cizonan; Germanium spiroamine; Fine crosslinking Spore-forming acid; triaminoquinone; 2,2',2”-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verrucosporin A, baculosporin A, and serpentin); urethane; vincristine; dacarbazine; mannomustine; dibromomannitol; dibromoeusol; piperbbromoethane; gacytosine; arabinoside (Ara-C); cyclophosphamide; taxanes, such as paclitaxel and docetaxel gemcitabine. 6-Thioguanine; mercaptopurine; platinum coordination complexes such as cisplatin, oxaliplatin, and carboplatin; vincristine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edaraxazole; daunorubicin; aminopterin; capecitabine; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); visual pigments such as retinoic acid; capecitabine; carboplatin, procarbazine, procamycin, gemcitabine, novibenten, farnesyltransferase inhibitors, farnesyl-transferase inhibitors, transplatinum, and one or more pharmaceutically acceptable salts, acids, or derivatives of any of the above substances.

[0076] In some implementations, the drugs used to treat lower limb ischemia include, but are not limited to, vasodilators such as sargrelate.

[0077] The ninth aspect of the present invention provides any of the following applications: (1) The use of the cells, exosomes, drugs, or drug compositions described in the eighth aspect of the present invention in the preparation of a drug for treating vascular aging or vascular aging-related diseases or conditions; (2) Application of NNMT promoters in the preparation of drugs for treating vascular aging or vascular aging-related diseases or conditions; (3) Application of NNMT promoters in the preparation of drugs that protect or improve vascular endothelial function and integrity, enhance vascular endothelial barrier function, improve vascular contractile tension, have anti-inflammatory and anti-oxidative stress effects, reverse fibrosis, and treat type 2 diabetes, obesity, and metabolic syndrome. (4) Application of Codonopsis pilosula cyclic peptide B as an NNMT promoter; (5) Application of reagents for detecting NNMT in the preparation of products that predict the sensitivity of vascular aging or vascular aging-related diseases to Codonopsis pilosula cyclic peptide B; (6) The application of the system and apparatus described in the fifth aspect (1) of the present invention in the production of cells expressing high levels of NNMT; (7) The application of the system and apparatus described in the fifth aspect (2) of the present invention in the production of exosomes rich in NNMT; (8) The application of the system and apparatus described in the fifth aspect (3) of the present invention in screening drugs that affect the enhancing effect of Codonopsis pilosula cyclic peptide B on NNMT expression; (9) The use of the system and apparatus described in the fifth aspect (4) of the present invention in screening drugs for treating vascular aging or vascular aging-related diseases or conditions; (10) The application of the system and apparatus described in the fifth aspect (5) of the present invention in predicting the sensitivity of vascular aging or vascular aging-related diseases or conditions to Codonopsis pilosula cyclic peptide B drug.

[0078] In some implementations, the diseases or conditions related to vascular aging include, but are not limited to, atherosclerosis, hypertension, stroke, tumors, lower limb ischemia, and diabetic nephropathy.

[0079] In some implementations, the vascular aging or vascular aging-related diseases or conditions are selected from lower limb ischemia, diabetic nephropathy, etc.

[0080] In some embodiments, the NNMT promoter includes substances that promote NNMT activity, promote the stability of the NNMT gene or protein, promote NNMT expression levels, and promote the effective duration of NNMT action.

[0081] In some implementations, the effects of promoters can also be achieved through knock-in. Knock-in refers to the targeted insertion of a transgene into the host cell genome, resulting in transgene expression and / or altered expression of the endogenous target gene (e.g., increased (including ectopic) or decreased expression), for example, by introducing an additional copy of the target gene or by operatively inserting a regulatory sequence that provides an endogenous copy of the target gene to enhance expression. Knock-in transgenes can include heterozygous knock-in or homozygous knock-in. Knock-in also encompasses conditional knock-in, where transgene expression and / or altered expression of the endogenous target gene can occur, for example, by exposing the animal to a substance that promotes such expression, by introducing an enzyme that promotes recombination at the targeted insertion site, or by some other method used to alter the targeted insertion site.

[0082] In some embodiments, the promoter includes, but is not limited to, nucleic acid molecules, carbohydrates, liposomes, small molecule chemicals, antibody drugs, peptides, proteins, or reagents used in gene editing. In this invention, the term nucleic acid refers to polynucleotides such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). As equivalents, nucleic acids also include DNA or RNA analogs derived from nucleotide analogs and, where applicable, single-stranded (sense or antisense) and double-stranded polynucleotides. Liposomes refer to small vesicles composed of various types of lipids, phospholipids, and / or surfactants, which can be used for drug delivery to mammals. Antibodies cover complete monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two complete antibodies, and antibody fragments, provided they exhibit the desired antigen-binding activity and fall within the scope of this invention.

[0083] In some embodiments, the NNMT promoter is selected from substances that promote the expression level of NNMT.

[0084] In some embodiments, the substance that promotes the expression level of NNMT is selected from Codonopsis pilosula cyclic peptide B.

[0085] In some implementations, the NNMT promoter may also be a nucleic acid encoding the NNMT protein.

[0086] In some embodiments, the nucleic acid encoding the NNMT protein is DNA encoding the NNMT protein.

[0087] In some embodiments, nucleic acid molecules are introduced into cells using non-viral or virus-based methods. The nucleic acid molecule may be a sequence encoding the entire protein or a functional portion thereof. In some embodiments, the Gene ID of the DNA encoding the NNMT protein is NC_000011.10.

[0088] Non-viral methods include any suitable method that does not use viral DNA or viral particles as a delivery system to introduce nucleic acid molecules into cells. Exemplary non-viral methods include, but are not limited to, calcium phosphate transfection, liposome transfection, nuclear transfection, acoustic pore effect, heat shock transfection, magnetic transfection, and electroporation. For virus-based methods, any useful viral vector known in the art can be used in the methods described in this invention. Examples of viral vectors include, but are not limited to, retroviral vectors, adenovirus vectors, lentiviral vectors, and adeno-associated virus vectors. In some aspects, retroviral vectors are used to introduce said nucleic acid molecules into cells according to standard procedures known in the art.

[0089] The tenth aspect of the present invention provides a method for treating vascular aging or vascular aging-related diseases or conditions, the method comprising administering a therapeutically effective amount of Codonopsis pilosula cyclic peptide B, cells, exosomes, drugs, or drug compositions as described in the eighth aspect of the present invention.

[0090] The advantages and beneficial effects of this invention are as follows: This invention provides a method for preparing cells highly expressing NNMT, NNMT-rich exosomes, and their applications. This invention is the first to discover that Codonopsis pilosula cyclic peptide B can promote NNMT expression. Based on this, this invention provides a method for preparing cells highly expressing NNMT and a method for preparing NNMT-rich exosomes. Experiments have verified that the NNMT-rich exosomes can effectively treat vascular aging or vascular aging-related diseases or conditions. Attached Figure Description

[0091] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0092] Figure 1 This is a schematic flowchart of a drug screening method for screening drugs that affect the enhancing effect of Codonopsis pilosula cyclic peptide B on NNMT expression, provided by an embodiment of the present invention. Figure 2 This is a schematic diagram of a drug screening method provided in an embodiment of the present invention; Figure 3 This is a graph showing the experimental results of how Codonopsis pilosula cyclic peptide B enhances NNMT expression. Figure 4 This is a diagram showing the experimental results of Codonopsis pilosula cyclic peptide B alleviating lower limb ischemia-reperfusion injury; Figure 5 This is a diagram showing the experimental results of how Codonopsis pilosula cyclic peptide B improves vasomotor tension. Figure 6This is a diagram showing the experimental results of the effect of Codonopsis pilosula cyclic peptide B on the vasodilatory function of vascular smooth muscle. Figure 7 This is a graph showing the experimental results of how Codonopsis pilosula cyclic peptide B reduces inflammatory response; Figure 8 This is a diagram showing the experimental results of how Codonopsis pilosula cyclic peptide B resists oxidative stress. Figure 9 This is a schematic diagram of a drug screening system provided in an embodiment of the present invention for screening drugs that enhance the expression of NNMT by Codonopsis pilosula cyclic peptide B. Figure 10 This is a schematic diagram of a system for producing cells that highly express NNMT, provided in an embodiment of the present invention; Figure 11 This is a schematic diagram of a drug screening system provided in an embodiment of the present invention; Figure 12 These are experimental results showing how exosomes improve endothelial-dependent relaxation and promote perfusion recovery after hindlimb ischemia-reperfusion. Among them, Figures A and C show the successful entry and accumulation of NNMT inside the exosomes; Figure D shows the reactivation of the relaxation signaling pathway of senescent endothelial cells by exosomes; Figure E shows the relief of acute ischemia-reperfusion (I / R) injury by exosomes; Figure F shows the improvement of EF by exosomes; and Figure G shows the improvement of FS by exosomes. Detailed Implementation

[0093] To enable those skilled in the art to better understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0094] In some of the processes described in the specification, claims, and accompanying drawings of this invention, multiple operations appearing in a specific order are included. However, it should be clearly understood that these operations may not be executed in the order they appear herein, or may be executed in parallel. The operation numbers, such as 101, 102, etc., are merely used to distinguish different operations and do not represent any execution order. Furthermore, these processes may include more or fewer operations, and these operations may be executed sequentially or in parallel. It should be noted that the descriptions such as "first," "second," etc., in this document are used to distinguish different messages, devices, modules, etc., and do not represent a sequential order, nor do they limit "first" and "second" to different types.

[0095] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0096] Figure 1 This is a schematic flowchart of a drug screening method provided by an embodiment of the present invention for screening drugs that affect the enhancing effect of Codonopsis pilosula cyclic peptide B on NNMT expression, specifically including: S101: A mixture of cells treated with Codonopsis pilosula cyclic peptide B and test compounds; S102: Detection of NNMT expression levels in cells; S103: Compare the detected expression level results with the NNMT expression level obtained in the absence of the test compound to determine whether the test compound is a drug that promotes the enhancing effect of ginsenoside B on NNMT expression or a drug that inhibits the enhancing effect of ginsenoside B on NNMT expression.

[0097] In some embodiments, the determination method for the test compound being a drug that enhances the expression of NNMT by codonopsis pilosula cyclic peptide B is that the NNMT expression level detected after the addition of the test compound is higher than the NNMT expression level when the test compound is not present.

[0098] In some embodiments, the method for determining whether the test compound is a drug that inhibits the enhancing effect of codonopsis cyclopeptide B on NNMT expression is that the NNMT expression level detected after adding the test compound is lower than the NNMT expression level when the test compound is not present.

[0099] In some embodiments, the test compound does not directly affect NNMT expression in the absence of Codonopsis pilosula cyclic peptide B.

[0100] Figure 2 This is a schematic flowchart of a drug screening method provided in an embodiment of the present invention, specifically including: S201: Obtain NNMT initial expression level data in cells 1; S202: Mixed cells and test compounds; S203: Detect the expression level of NNMT in cells and obtain expression level data 2; S204: Compare expression level data 2 with expression level data 1. If expression level data 2 is higher than expression level data 1, then the test compound is a drug for treating vascular aging or vascular aging-related diseases or conditions.

[0101] In some implementations, the test compound is selected from proteins.

[0102] In some embodiments, the protein is a naturally occurring bioactive peptide, including but not limited to soybean peptides, corn peptides, wheat peptides, walnut peptides, pea peptides, and Codonopsis pilosula cyclic peptide B.

[0103] In some implementations, mixing the above-mentioned test compound with HUVECs cells increases the expression of NNMT by codonopsis pilosula cyclic peptide B.

[0104] In some implementation schemes, HUVECs were treated with HPB (Codonopsis pilosula cyclic peptide B) at concentrations of 10, 20, and 50, respectively. Cells were collected 24-48 hours after treatment to extract proteins for Western blotting (WB) experiments to observe NNMT expression. The results ( Figure 3 According to the results, compared with the Con group, the expression of NNMT in cells was increased at all concentrations.

[0105] In some implementations, the method further includes validating the functional activity of the screened drugs.

[0106] In some implementations, the method for verifying functional activity includes: The experimental animals were divided into three groups: the Con group (fed with physiological saline), the low-dose group (10 mg / kg), and the high-dose group (20 mg / kg). The relevant experiments were conducted 14 days after gavage.

[0107] Lower limb ischemia-reperfusion experiments were performed on all mice, and the results were as follows ( Figure 4 The results showed that HPB can effectively protect and improve vascular endothelial function and integrity, and enhance the barrier function and anti-inflammatory function of vascular endothelium, with particularly significant results in high-dose mice.

[0108] In some implementations, the method for verifying functional activity includes: In vitro vascular ring experiments on experimental animal blood vessels showed that HPB treatment improved vascular systolic tension in mice, with the low-dose group showing better improvement than the high-dose group. Figure 5 However, regarding the response of mouse blood vessels to SNPs, there was no significant difference in the effect of blood vessels on SNPs among the different groups. Figure 6 This demonstrates that HPB can specifically improve or protect endothelial function.

[0109] In some implementations, the method for verifying functional activity includes: The serum samples were analyzed using ELISA to detect the concentrations of IL-6 and MDA in each group. The results showed that, compared to the Con group, the inflammatory factors decreased after drug treatment, further validating its anti-inflammatory and antioxidant effects. Figures 7-8 ).

[0110] Figure 9 This is a schematic diagram of a drug screening system provided in an embodiment of the present invention for screening drugs that affect the enhancing effect of Codonopsis pilosula cyclic peptide B on NNMT expression, specifically including: 301: Mixing Unit: Mixing cells treated with Codonopsis pilosula cyclic peptide B with the test compound; 302: Detection unit: Detects the expression level of NNMT in cells; 303: Drug Screening Unit: Compares the detected expression level results with the NNMT expression level obtained in the absence of the test compound to determine whether the test compound is a drug that promotes the enhancing effect of ginsenoside B on NNMT expression or a drug that inhibits the enhancing effect of ginsenoside B on NNMT expression.

[0111] Figure 10 This is a schematic diagram of a system for producing cells that highly express NNMT, provided in an embodiment of the present invention, specifically including: 401: Mixed Unit: Mixed cells with Codonopsis pilosula cyclic peptide B; 402: Culture unit: Cultured mixed cells.

[0112] Figure 11 This invention provides a drug screening system, specifically comprising: 501: Data Acquisition Unit: Acquires NNMT initial expression level data in cells 1; 502: Mixing Unit: Mixing cells with test compounds; 503: Detection unit: Detects the expression level of NNMT in cells and obtains expression level data 2; 504: Drug screening unit: compare expression level data 2 with expression level data 1. If expression level data 2 is higher than expression level data 1, then the test compound is a drug for treating vascular aging or vascular aging-related diseases or conditions.

[0113] The present invention will be further described below with reference to embodiments. The following description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make equivalent modifications to the disclosed technical content to create equivalent embodiments. Any simple modifications or equivalent changes made to the following embodiments based on the technical essence of the present invention without departing from the scope of the invention are all within the protection scope of the present invention.

[0114] Example 1: Preparation of cells expressing high levels of NNMT and verification of the function of Codonopsis pilosula cyclic peptide B HUVECs with 2×10 4 -4×10 4 Cells were seeded at a density of 10 μM / cm² and cultured in a cell culture incubator. When the cell confluence reached 80%-90%, they were treated with 10 μM, 20 μM, and 50 μM HPB (Codonopsis pilosula cyclic peptide B) respectively, and cultured for another 24-48 hours. Cells were then harvested. Proteins were extracted and subjected to Western blotting (WB) to observe NNMT expression. The results (…) Figure 3According to the results, compared with the Con group, the expression of NNMT in cells was increased at all concentrations.

[0115] The experimental animals were divided into three groups: the Con group (fed with physiological saline), the low-dose group (10 mg / kg), and the high-dose group (20 mg / kg). The relevant experiments were conducted 14 days after gavage.

[0116] Lower limb ischemia-reperfusion experiments were performed on all mice, and the results were as follows ( Figure 4 The results showed that HPB can effectively protect and improve vascular endothelial function and integrity, and enhance the barrier function and anti-inflammatory function of vascular endothelium, with particularly significant results in high-dose mice.

[0117] In vitro vascular ring experiments on experimental animal blood vessels showed that HPB treatment improved vascular systolic tension in mice, with the low-dose group showing better improvement than the high-dose group. Figure 5 However, regarding the response of mouse blood vessels to SNPs, there was no significant difference in the effect of blood vessels on SNPs among the different groups. Figure 6 This demonstrates that HPB can specifically improve or protect endothelial function.

[0118] The serum samples were analyzed using ELISA to detect the concentrations of IL-6 and MDA in each group. The results showed that, compared to the Con group, the inflammatory factors decreased after drug treatment, further validating the anti-inflammatory and oxidative stress-resisting effects of Codonopsis pilosula cyclic peptide B. Figures 7-8 ).

[0119] Example 2: Exosome preparation, therapeutic uses, and functional verification Exosomes were extracted from the cells obtained above. The exosomes were separated and purified using ultracentrifugation. First, low-speed centrifugation (300xg, 2000xg) was performed to remove cells and cell debris, followed by ultracentrifugation (100,000-120,000xg) to precipitate the exosomes. The extracts were observed under a transmission electron microscope, revealing cup-shaped spherical or biconcave disc-shaped structures with diameters approximately 30-150 nm. The samples were well-dispersed without severe aggregation. Western blotting was used to identify the extracts, detecting the expression of exosome marker proteins (CD9, CD63, CD81, etc.) and NNMT. The extraction results from different groups showed that the NNMT protein content in the exosomes of the drug intervention group was significantly higher than that of the control group, and the higher the concentration of the intervention drug, the higher the content of secreted exosomes.

[0120] Exosomes were isolated using a superposition method, and the integrity of the carrier was confirmed by the typical cup-shaped structure observed under electron microscopy and the presence of the markers CD63 / CD9. Western blotting confirmed that NNMT successfully entered and accumulated within the exosomes, forming a 'bionanomedicine'. Figure 12AC).

[0121] Exosomes rich in NNMT were prepared, and the protein concentration was determined to be 1.0 mg / mL by the BCA method. 18-month-old senescent mice were injected intravenously with 100 μL of the above exosome solution per mouse (containing 100 μg of exosome protein). Injections were given every 3 days for 4 weeks. After this intervention, a significant improvement in the ACh-induced diastolic response (ACh-EDR) was observed by vascular ring assay. This indicates that exosomes can cross the circulatory system and reactivate the diastolic signaling pathway in senescent endothelial cells by supplementing endothelial NNMT levels. Figure 12 D). In experiments simulating clinical acute ischemia-reperfusion (I / R), this exosome demonstrated significant anti-damage potential, which not only validated its long-term anti-aging value but also revealed its application prospects in the management of acute vascular events. Figure 12 E). To further evaluate the intervention effect of NNMT-rich exosomes on systemic aging, we performed real-time echocardiographic monitoring of cardiac function in 18-month-old naturally aging mice. Mice in the experimental group were injected via tail vein with HUVEC-derived (HP-B treated) NNMT-rich exosomes, while the control group received an equal amount of ordinary exosomes. The results showed that, compared with the control group, aging mice treated with NNMT-rich exosomes exhibited significant improvements in ventricular systolic performance: Ejection Fraction (EF): The EF value of the experimental group mice was significantly higher than that of the control group ( Figure 12 F). Furthermore, fractional shortening (FS): The FS values ​​of the experimental group mice also showed a synchronous increase (F). Figure 12 (G) The improvement in FS reflects the improvement in left ventricular radial contractile capacity, suggesting that myocardial fiber contractile compliance tends to be younger under drug intervention. These data strongly demonstrate that NNMT-rich exosomes have systemic benefits beyond local vascular repair. The improvement in cardiac EF and FS, together with the previously observed improvement in acetylcholine-dependent vasodilation, constitutes a complete protective chain: namely, reducing peripheral resistance (afterload) by optimizing endothelial function and possibly improving myocardial metabolism through direct nutritional support, thereby reversing age-related cardiovascular function decline. The above experiments suggest that it can improve endothelial-dependent vasodilation and promote perfusion recovery after hindlimb ischemia-reperfusion.

[0122] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A method for preparing cells highly expressing NNMT, characterized in that, The method includes mixing Codonopsis pilosula cyclic peptide B and / or Codonopsis pilosula extract, as well as cells.

2. The preparation method according to claim 1, characterized in that, The concentration of the Codonopsis pilosula cyclic peptide B used is 1 nM-100 μM; Preferably, the concentration of the ginseng cyclic peptide B is selected from 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 20 μM, 50 μM, and 100 μM; Preferably, the effective concentration of ginseng cyclic peptide B in the ginseng extract is 1 nM-100 μM; Preferably, the effective concentration of Pseudostellaria heterophylla cyclic peptide B in the Pseudostellaria heterophylla extract is selected from 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 20 μM, 50 μM, and 100 μM; Preferably, the mixing time is 0.5-96 hours; Preferably, the mixing time is 24-48 hours; Preferably, when the cell confluence rate reaches 80%-90%, the mixture of Codonopsis pilosula cyclic peptide B and / or Codonopsis pilosula extract, along with the cells, is added. Preferably, the cells include vascular endothelial cells; Preferably, the vascular endothelial cells include HUAECs, HCAECs, HAAECs, HDMECs, HBMECs, HRMECs, HPMECs, and HIMECs. Preferably, the cells are selected from HUVECs cells.

3. A method for preparing exosomes rich in NNMT, characterized in that, The preparation method includes collecting exosomes generated from cells prepared by the preparation method according to any one of claims 1-2; Preferably, the collection method includes ultracentrifugation, density gradient centrifugation, size exclusion chromatography, polymer precipitation, immunomagnetic bead method, and ultrafiltration. Preferably, the collection method is selected from ultracentrifugation and size exclusion chromatography.

4. A drug screening method for detecting the enhancing effect of Codonopsis pilosula cyclic peptide B on NNMT expression, characterized in that, The drug screening method includes: Mixed cells that had been treated with Codonopsis pilosula cyclic peptide B with test compounds; Detect the expression level of NNMT in cells; The detected expression level results were compared with the NNMT expression level obtained in the absence of the test compound to determine whether the test compound was a drug that promoted the enhancing effect of ginsenoside B on NNMT expression or a drug that inhibited the enhancing effect of ginsenoside B on NNMT expression. Preferably, the method for determining whether the test compound is a drug that enhances the expression of NNMT by codonopsis pilosula cyclic peptide B is that the NNMT expression level detected after adding the test compound is higher than the NNMT expression level when the test compound is not present. Preferably, the method for determining whether the test compound is a drug that inhibits the enhancing effect of Codonopsis pilosula cyclic peptide B on NNMT expression is that the NNMT expression level detected after adding the test compound is lower than the NNMT expression level when the test compound is not present. Preferably, the compound includes small molecule compounds and macromolecule compounds; Preferably, the macromolecular compound includes proteins, DNA, and RNA.

5. Any one of the following methods: (1) A method for regulating NNMT expression in in vitro cells, characterized in that, The method includes mixing cells and Codonopsis pilosula cyclic peptide B; (2) A drug screening method, characterized in that the screening method is based on NNMT, and the drug is a drug for treating vascular aging or vascular aging-related diseases or conditions, specifically including: Obtain NNMT initiation expression level data in cells 1; Mixing cells and test compounds; The expression level of NNMT in cells was detected, and expression level data were obtained. Compare expression level data 2 with expression level data 1. If expression level data 2 is higher than expression level data 1, then the test compound is a drug for treating vascular aging or vascular aging-related diseases or conditions. Preferably, the method further includes culturing the mixed cells; (3) A method for predicting the sensitivity of vascular aging or vascular aging-related diseases or conditions to Codonopsis pilosula cyclic peptide B, characterized in that the method includes detecting the expression level of NNMT after administration of Codonopsis pilosula cyclic peptide B; Preferably, the cells include vascular endothelial cells; Preferably, the vascular endothelial cells include, but are not limited to, HUAECs, HCAECs, HAAECs, HDMECs, HBMECs, HRMECs, HPMECs, and HIMECs. Preferably, the cells are selected from HUVECs cells; Preferably, the vascular aging-related diseases or conditions include atherosclerosis, hypertension, stroke, tumors, lower limb ischemia, and diabetic nephropathy; Preferably, the vascular aging-related diseases or conditions are selected from lower limb ischemia and diabetic nephropathy.

6. Any one of the following products: (1) A system and apparatus for producing cells that highly express NNMT, characterized in that, The system and apparatus include: Mixed unit: Mixed cells with Codonopsis pilosula cyclic peptide B; Culture unit: culturing the mixed cells; (2) A system or apparatus for producing exosomes rich in NNMT, characterized in that the system or apparatus comprises: Cell acquisition unit: Acquires cells prepared by the preparation method according to any one of claims 1-2; Collection unit: collects exosomes produced by cells prepared by the preparation method according to any one of claims 1-2; (3) A drug screening system and apparatus for screening drugs that enhance the expression of NNMT by Codonopsis pilosula cyclic peptide B, characterized in that the system and apparatus comprise: Mixing unit: Mixing cells treated with Codonopsis pilosula cyclic peptide B with the test compound; Detection unit: Detects the expression level of NNMT in cells; Drug screening unit: The detected expression level results are compared with the NNMT expression level obtained in the absence of the test compound to identify the test compound as either a drug that promotes the enhancement of NNMT expression by ginseng cyclic peptide B or a drug that inhibits the enhancement of NNMT expression by ginseng cyclic peptide B. Preferably, the method for determining whether the test compound is a drug that enhances the expression of NNMT by codonopsis pilosula cyclic peptide B is that the NNMT expression level detected after adding the test compound is higher than the NNMT expression level when the test compound is not present. Preferably, the method for determining whether the test compound is a drug that inhibits the enhancing effect of Codonopsis pilosula cyclic peptide B on NNMT expression is that the NNMT expression level detected after adding the test compound is lower than the NNMT expression level when the test compound is not present. (4) A drug screening system or apparatus, characterized in that the drug is a drug for treating vascular aging or vascular aging-related diseases or symptoms, specifically including: Data acquisition unit: Acquire NNMT initial expression level data of cells 1; Mixing unit: mixing cells with test compounds; Detection unit: Detects the expression level of NNMT in cells and obtains expression level data 2; Drug screening unit: compare expression level data 2 with expression level data 1. If expression level data 2 is higher than expression level data 1, then the test compound is a drug for treating vascular aging or vascular aging-related diseases or conditions. (5) A system or apparatus for predicting the sensitivity of vascular aging or vascular aging-related diseases or conditions to Codonopsis pilosula cyclic peptide B, characterized in that the system or apparatus specifically comprises: Acquisition Unit: Acquire NNMT initial expression level data of cells 1; Drug administration unit: administration of Codonopsis pilosula cyclic peptide B; Detection unit: Detect the expression level of NNMT after administration of Codonopsis pilosula cyclic peptide B, and obtain expression level data 2; Prediction Unit: Compares expression level data 2 with expression level data 1. If expression level data 2 is higher than expression level data 1, it is predicted that the vascular aging or vascular aging-related diseases or conditions are highly sensitive to Codonopsis pilosula cyclic peptide B. If expression level data 2 is not higher than expression level data 1, it is predicted that the vascular aging or vascular aging-related diseases or conditions are less sensitive to Codonopsis pilosula cyclic peptide B. Preferably, the cells include vascular endothelial cells; Preferably, the vascular endothelial cells include, but are not limited to, HUAECs, HCAECs, HAAECs, HDMECs, HBMECs, HRMECs, HPMECs, and HIMECs. Preferably, the cells are selected from HUVECs cells; Preferably, the vascular aging-related diseases or conditions include atherosclerosis, hypertension, stroke, tumors, lower limb ischemia, and diabetic nephropathy; Preferably, the vascular aging-related diseases or conditions are selected from lower limb ischemia and diabetic nephropathy.

7. A computer device, characterized in that, include: A memory and a processor, wherein the memory is used to store program instructions; and the processor is used to invoke the program instructions, which, when executed, implement the preparation method according to any one of claims 1-2, the preparation method according to claim 3, the drug screening method according to claim 4, the drug screening method according to claim 5, or the prediction method according to claim 5.

8. A computer-readable storage medium having a computer program thereon, comprising: When the computer program is executed by the processor, it implements the preparation method according to any one of claims 1-2, the preparation method according to claim 3, the drug screening method according to claim 4, the drug screening method according to claim 5, or the prediction method according to claim 5.

9. Any one of the following products: (1) A cell that highly expresses NNMT, characterized in that, The cells are prepared by the preparation method according to any one of claims 1-2; (2) An exosome rich in NNMT, characterized in that the exosome is prepared by the preparation method of claim 3; (3) A drug obtained based on the drug screening method of claim 4 or the drug screening method of claim 5; (4) A pharmaceutical composition, characterized in that the pharmaceutical composition comprises cells expressing NNMT as described in claim 9 (1), NNMT-rich exosomes as described in claim 9 (2), or the drug as described in claim 9 (3); Preferably, the pharmaceutical composition further includes a pharmaceutically acceptable carrier and / or excipients.

10. Any of the following applications: (1) The use of the cell, exosome, drug, or drug composition of claim 9 in the preparation of a medicament for treating vascular aging or vascular aging-related diseases or conditions; (2) Application of NNMT promoters in the preparation of drugs for treating vascular aging or vascular aging-related diseases or conditions; (3) Application of NNMT promoters in the preparation of drugs that protect or improve vascular endothelial function and integrity, enhance vascular endothelial barrier function, improve vascular contractile tension, have anti-inflammatory and anti-oxidative stress effects, reverse fibrosis, and treat type 2 diabetes, obesity, and metabolic syndrome. (4) Application of Codonopsis pilosula cyclic peptide B as an NNMT promoter; (5) Application of reagents for detecting NNMT in the preparation of products that predict the sensitivity of vascular aging or vascular aging-related diseases to Codonopsis pilosula cyclic peptide B; (6) The application of the system or apparatus described in claim 6(1) in the production of cells expressing high levels of NNMT; (7) The application of the system or apparatus described in claim 6(2) in the production of exosomes rich in NNMT; (8) The use of the system and apparatus described in claim 6 (3) in screening drugs that affect the enhancing effect of Codonopsis pilosula cyclic peptide B on NNMT expression; (9) The use of the system or apparatus described in claim 6 (4) in screening drugs for treating vascular aging or vascular aging-related diseases or conditions; (10) The use of the system or apparatus described in claim 6 (5) in predicting the sensitivity of vascular aging or vascular aging-related diseases or conditions to Codonopsis pilosula cyclic peptide B drug; Preferably, the vascular aging-related diseases or conditions include atherosclerosis, hypertension, stroke, tumors, lower limb ischemia, and diabetic nephropathy; Preferably, the vascular aging-related diseases or conditions are selected from lower limb ischemia and diabetic nephropathy; Preferably, the NNMT promoter includes substances that promote NNMT activity, promote the stability of the NNMT gene or protein, promote the expression level of NNMT, and promote the effective duration of NNMT action. Preferably, the NNMT promoter is selected from substances that promote the expression level of NNMT; Preferably, the substance that promotes the expression level of NNMT is selected from Codonopsis pilosula cyclic peptide B; Preferably, the reagent is selected from oligonucleotide probes that specifically recognize the NNMT gene, primers that specifically amplify the NNMT gene, or binding agents that specifically bind to the protein encoded by the NNMT gene. Preferably, the reagent further includes a detectable marker; Preferably, the detectable markers include DNA barcodes, radioactive isotopes, nucleotide chromophores, enzymes, substrates, fluorescent molecules, chemiluminescent components, magnetic particles, and bioluminescent components; Preferably, the product includes a chip, a test strip, or a nucleic acid membrane strip; Preferably, the chip includes a gene chip or a protein chip.

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