CN115252759A - 酸奶衍生多肽在制备延缓端粒缩短药物及抗衰老药物中的应用 - Google Patents
酸奶衍生多肽在制备延缓端粒缩短药物及抗衰老药物中的应用 Download PDFInfo
- Publication number
- CN115252759A CN115252759A CN202211021600.7A CN202211021600A CN115252759A CN 115252759 A CN115252759 A CN 115252759A CN 202211021600 A CN202211021600 A CN 202211021600A CN 115252759 A CN115252759 A CN 115252759A
- Authority
- CN
- China
- Prior art keywords
- yoghourt
- aging
- derived
- polypeptide
- delaying
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 65
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 62
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 58
- 108091035539 telomere Proteins 0.000 title claims abstract description 46
- 102000055501 telomere Human genes 0.000 title claims abstract description 46
- 210000003411 telomere Anatomy 0.000 title claims abstract description 46
- 235000013618 yogurt Nutrition 0.000 title claims abstract description 45
- 239000003814 drug Substances 0.000 title claims abstract description 32
- 230000003712 anti-aging effect Effects 0.000 title claims abstract description 15
- 238000004904 shortening Methods 0.000 title claims abstract description 15
- 229940079593 drug Drugs 0.000 claims abstract description 13
- 238000002360 preparation method Methods 0.000 claims description 9
- 239000002775 capsule Substances 0.000 claims description 4
- 239000008187 granular material Substances 0.000 claims description 4
- 239000007924 injection Substances 0.000 claims description 4
- 238000002347 injection Methods 0.000 claims description 4
- 239000003826 tablet Substances 0.000 claims description 4
- 239000003795 chemical substances by application Substances 0.000 claims description 3
- 238000013268 sustained release Methods 0.000 claims description 3
- 239000012730 sustained-release form Substances 0.000 claims description 3
- 239000003405 delayed action preparation Substances 0.000 claims 1
- 230000032683 aging Effects 0.000 abstract description 14
- 210000004027 cell Anatomy 0.000 description 33
- 230000000694 effects Effects 0.000 description 21
- 238000011282 treatment Methods 0.000 description 11
- 230000036542 oxidative stress Effects 0.000 description 10
- 230000006907 apoptotic process Effects 0.000 description 9
- 230000003078 antioxidant effect Effects 0.000 description 8
- 241000699666 Mus <mouse, genus> Species 0.000 description 7
- 239000003963 antioxidant agent Substances 0.000 description 7
- 230000010094 cellular senescence Effects 0.000 description 7
- 238000000034 method Methods 0.000 description 7
- 230000009758 senescence Effects 0.000 description 7
- 239000000243 solution Substances 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- 108010005774 beta-Galactosidase Proteins 0.000 description 5
- 229940088598 enzyme Drugs 0.000 description 5
- 230000001965 increasing effect Effects 0.000 description 5
- 239000013641 positive control Substances 0.000 description 5
- GVJHHUAWPYXKBD-UHFFFAOYSA-N (±)-α-Tocopherol Chemical compound OC1=C(C)C(C)=C2OC(CCCC(C)CCCC(C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-UHFFFAOYSA-N 0.000 description 4
- 102000009092 Proto-Oncogene Proteins c-myc Human genes 0.000 description 4
- 108010087705 Proto-Oncogene Proteins c-myc Proteins 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 230000002829 reductive effect Effects 0.000 description 4
- 230000004083 survival effect Effects 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 3
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 241000699670 Mus sp. Species 0.000 description 3
- 238000005299 abrasion Methods 0.000 description 3
- 235000006708 antioxidants Nutrition 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 102000005936 beta-Galactosidase Human genes 0.000 description 3
- 210000000349 chromosome Anatomy 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 239000002207 metabolite Substances 0.000 description 3
- 210000005259 peripheral blood Anatomy 0.000 description 3
- 239000011886 peripheral blood Substances 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 102000007469 Actins Human genes 0.000 description 2
- 108010085238 Actins Proteins 0.000 description 2
- 238000007400 DNA extraction Methods 0.000 description 2
- 238000012449 Kunming mouse Methods 0.000 description 2
- 101710135898 Myc proto-oncogene protein Proteins 0.000 description 2
- 102100038895 Myc proto-oncogene protein Human genes 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 238000011529 RT qPCR Methods 0.000 description 2
- 108010017842 Telomerase Proteins 0.000 description 2
- 101710150448 Transcriptional regulator Myc Proteins 0.000 description 2
- 229930003427 Vitamin E Natural products 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- WQZGKKKJIJFFOK-FPRJBGLDSA-N beta-D-galactose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-FPRJBGLDSA-N 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 239000006143 cell culture medium Substances 0.000 description 2
- 230000003833 cell viability Effects 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 239000002552 dosage form Substances 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- WIGCFUFOHFEKBI-UHFFFAOYSA-N gamma-tocopherol Natural products CC(C)CCCC(C)CCCC(C)CCCC1CCC2C(C)C(O)C(C)C(C)C2O1 WIGCFUFOHFEKBI-UHFFFAOYSA-N 0.000 description 2
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 2
- 230000002427 irreversible effect Effects 0.000 description 2
- 210000000265 leukocyte Anatomy 0.000 description 2
- 210000005228 liver tissue Anatomy 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 239000008267 milk Substances 0.000 description 2
- 235000013336 milk Nutrition 0.000 description 2
- 210000004080 milk Anatomy 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000010172 mouse model Methods 0.000 description 2
- 230000004792 oxidative damage Effects 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 235000019165 vitamin E Nutrition 0.000 description 2
- 229940046009 vitamin E Drugs 0.000 description 2
- 239000011709 vitamin E Substances 0.000 description 2
- WQZGKKKJIJFFOK-SVZMEOIVSA-N (+)-Galactose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-SVZMEOIVSA-N 0.000 description 1
- 102000000412 Annexin Human genes 0.000 description 1
- 108050008874 Annexin Proteins 0.000 description 1
- 238000009010 Bradford assay Methods 0.000 description 1
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 1
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 229920002449 FKM Polymers 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 108091081062 Repeated sequence (DNA) Proteins 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 102100032938 Telomerase reverse transcriptase Human genes 0.000 description 1
- 102000010823 Telomere-Binding Proteins Human genes 0.000 description 1
- 108010038599 Telomere-Binding Proteins Proteins 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 230000003044 adaptive effect Effects 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000006944 antioxidant bioactivity Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 210000000748 cardiovascular system Anatomy 0.000 description 1
- 230000032677 cell aging Effects 0.000 description 1
- 230000032823 cell division Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000004148 curcumin Substances 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000007407 health benefit Effects 0.000 description 1
- 230000007124 immune defense Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000007413 intestinal health Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- PGSADBUBUOPOJS-UHFFFAOYSA-N neutral red Chemical compound Cl.C1=C(C)C(N)=CC2=NC3=CC(N(C)C)=CC=C3N=C21 PGSADBUBUOPOJS-UHFFFAOYSA-N 0.000 description 1
- 150000007523 nucleic acids Chemical group 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000008844 regulatory mechanism Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000010187 selection method Methods 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000007447 staining method Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 229960004793 sucrose Drugs 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 238000009423 ventilation Methods 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/08—Peptides having 5 to 11 amino acids
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Engineering & Computer Science (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Toxicology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
本发明属于生物技术及生物制药技术领域，公开了一种酸奶衍生多肽在制备延缓端粒缩短药物及抗衰老药物中的应用。本发明发掘了酸奶衍生多肽的医疗新用途，开拓了一个新的应用领域，有望成为延缓端粒缩短和抗衰老的有效药物，同事为开发和扩展酸奶衍生多肽的抗衰老提供了基础。
Description
技术领域
本发明属于生物技术及生物制药技术领域，涉及酸奶衍生多肽在制备延缓端粒缩短药物及抗衰老药物中的应用。
背景技术
端粒是存在于真核细胞线状染色体末端的DNA-蛋白质复合体，它与端粒结合蛋白一起构成了染色体特殊的“帽子”结构，其主要功能是保持染色体的完整性使其免受损伤。随着细胞的分裂，端粒会进行性缩短，而当端粒缩短至一定的阈值时，细胞会发出损伤信号，并启动细胞衰老和凋亡程序，从而使得细胞走向不可逆性衰老，进而死亡，因此，端粒长度的维持对细胞衰老乃至个体衰老至关重要。经典的“端粒学说”表明，氧化应激能够加剧端粒磨损：一方面，富含鸟嘌呤的端粒DNA序列易成为急性氧化损伤的靶点；另一方面，活性氧等氧化应激产物可通过破坏端粒长度调节机制从而加速端粒磨损。因此，细胞内氧化损伤累积可能是端粒磨损的重要原因。此外，端粒酶逆转录酶活性是端粒长度的另一重要调节因子，通过利用自身RNA模板功能和逆转录酶活性合成新的端粒DNA重复序列，从而在细胞增殖的过程中参与端粒长度的维持。
酸奶由牛奶经微生物发酵而来，发酵过程为酸奶提供各类微生物活性代谢产物的同时也引入了多种具有预防和保健功能的活性肽，这些活性肽为心血管系统、神经系统、肠道健康和免疫防御等方面提供了多重的健康益处。此外，酸奶中的活性肽在胃肠消化阶段能够进一步释放出生物学活性更高的小分子衍生多肽。然而，酸奶中的活性肽能否通过抗氧化作用延缓端粒长度的磨损速度尚不明确。
发明内容
针对氧化应激加速端粒缩短进而导致细胞不可逆衰老的技术问题，本发明目的旨在提供酸奶衍生多肽在制备延缓端粒药物中的应用。
经研究发现，酸奶可通过增强抗氧化能力而延缓衰老模型小鼠端粒长度的磨损速度。通过与牛奶经消化后的多肽种类进行对比，筛选出酸奶中特有的具有潜在抗氧化生物活性的低分子量衍生肽，其序列分别为PLGTQ(脯氨酸-亮氨酸-甘氨酸-苏氨酸-谷氨酰胺)和VLNPW(缬氨酸-亮氨酸-天冬酰胺-脯氨酸-色氨酸)，具体筛选过程参见(Peptideprofiling and the bioactivity character of yogurt in the simulatedgastrointestinal digestion,Journal of Proteomics,Volume 141,1June 2016,Pages24-46)。
编码PLGTQ和VLNPW的核酸序列如下所示：
PLGTQ:ccgctgggcacccag
VLNPW:gtgctgaacccgtgg
本发明提供了酸奶衍生多肽在制备延缓端粒缩短药物中的应用。所述酸奶衍生多肽为具有序列PLGTQ或VLNPW的多肽。本发明提供的酸奶衍生多肽在制备延缓端粒缩短药物中的应用，对药物剂型和制备方法没有特别限制，可采用本领域常规通用的制备方法制成片剂、胶囊、颗粒剂、缓释剂、注射剂等各种剂型。
鉴于上述酸奶衍生多肽能够有效延缓端粒缩短，进而延缓衰老和死亡，本发明进一步提供了上述酸奶衍生多肽在制备抗衰老药物中的应用。本发明提供的酸奶衍生多肽在制备抗衰老药物中的应用，对药物剂型和制备方法没有特别限制，可采用本领域常规通用的制备方法制成片剂、胶囊、颗粒剂、缓释剂、注射剂等各种剂型。
与现有技术相比，本发明提供的酸奶衍生多肽在制备延缓端粒缩短药物及抗衰老药物中的应用具有以下有益效果：
(1)本发明发掘了酸奶衍生多肽的医疗新用途，开拓了一个新的应用领域，有望成为延缓端粒缩短和抗衰老的有效药物，同事为开发和扩展酸奶衍生多肽的抗衰老提供了基础。
(2)酸奶衍生多肽(例如具有序列PLGTQ和VLNPW的多肽)能够通过调节c-Myc/TERT信号通路和氧化应激水平，进而延缓细胞端粒磨损速度。
(3)酸奶衍生多肽(例如具有序列PLGTQ和VLNPW的多肽)能够降低β-半乳糖苷酶活性，提高抗氧化能力，进而延缓端粒磨损速度，从而发挥抗衰老作用。
附图说明
为了更清楚地说明本发明实施例或现有技术中的方案，以下将对实施例或现有技术描述中所需要使用的附图作简单介绍。
图1为不同条件下衰老小鼠外周血白细胞端粒长度测试结果。
图2为不同条件下衰老小鼠肝脏β-gal活性测试结果。
图3为CCK8法检测酸奶衍生多肽对细胞衰老模型细胞活力的测试结果。
图4为不同条件下细胞衰老模型相对端粒长度的测试结果。
图5为不同条件下细胞衰老模型抗氧化酶(GSH-Px、CAT和SOD活性)和氧化应激代谢产物(ROS水平和MDA水平)的影响。
图6为不同条件下细胞衰老模型凋亡率的测试结果。
图7为不同条件下细胞衰老模型TERT和c-Myc蛋白表达的测试结果；其中β-actin(肌动蛋白)作为内参。
具体实施方式
以将结合附图对本发明各实施例的技术方案进行清楚、完整的描述，显然，所描述实施例仅仅是本发明的一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动的前提下所得到的所有其它实施例，都属于本发明。
具有序列PLGTQ和VLNPW的酸奶衍生多肽采用常规技术手段得到，参见(Peptideprofiling and the bioactivity character of yogurt in thesimulatedgastrointestinal digestion,Journal of Proteomics,Volume 141,1 June2016,Pages 24-46)。
实施例1—qPCR法检测酸奶衍生多肽对小鼠端粒长度的影响
实验材料：昆明小鼠(SPF级)，雄性，3月龄，购于北京维通利华实验动物公司。
实施步骤：
(1)动物饲养及分组：
将80只5周龄雄性昆明小鼠随机分为空白对照组，衰老模型组、阳性对照组、PLGTQ和VLNPW。除空白对照组外，其余四组均进行建模处理。
建模处理：动物于温度20～25℃，相对湿度65～70％，明暗交替周期为12h，通风良好的环境中饲养。适应性喂养一周后，通过持续6周皮下注射80mg/kg D-gal制备衰老小鼠模型，第6周末，小鼠出现反应迟缓，活动量减少，毛发枯黄无光泽，精神萎靡等现象，表明衰老模型构建成功。
待模型构建成功后，给予阳性对照组(维生素E 100mg/kg/天)、PLGTQ和VLNPW(20mg/kg/天)持续8周每日灌胃相应剂量的药物，对照组灌胃无菌生理盐水(20mg/kg/天)。
(2)端粒长度检测：
按DNA提取试剂盒说明书分别提取各组小鼠外周血白细胞DNA后，以36B4为内参进行PCR扩增检测相对端粒长度，每个样本重复检测三次，循环条件为95℃预变性2min，95℃变性5s，60℃退火和延伸10s，进行40个循环，采用
法计算基因相对表达量，引物序列见表1。
表1引物名称和序列
实验结果如图1所示，与模型组相比，阳性对照组、PLGTQ和VLNPW组端粒长度显著延长，且差异均具有统计学意义，表明两种多肽与抗衰老阳性对照维生素E一样可以显著缓解衰老模型小鼠外周血端粒长度的磨损速度。
实施例2—染色法检测小鼠肝组织β-半乳糖酶活性
实施步骤：按照实施例1设置动物分组，进行药物处理。剖取药物干预后的各组小鼠肝脏组织，利用多聚甲醛固定24h，以蔗糖浸泡过夜后制备冰冻切片，经孵育液显色过夜，中性红复染后于油镜下观察各组切片蓝色阳性信号颗粒数量。
实验结果如图2所示，与模型组相比，阳性对照组、PLGTQ和VLNPW组蓝色信号颗粒数量显著减少，表明β-gal活性减弱，提示两种多肽与维生素E具有相同的缓解模型小鼠衰老的作用。
实施例3—CCK8法检测酸奶衍生多肽对细胞衰老模型细胞活力的影响
实验材料：LO2细胞购于上海中科院典藏细胞库
实施步骤：将处于对数期的LO2细胞接种于96孔板中，细胞密度为5×103个/孔，每组设置6个重复孔，待细胞贴壁后按照不同实验分组要求给予相应的干预处理，空白组是不含任何细胞的DMEM完全培养基；对照组是不含多肽的细胞培养液(DMEM完全培养基作为培养液)；实验组是分别加入0.01、0.05、0.1、0.5、1、5、10、50和100μg/ml多肽的细胞培养液。
分别干预24h后去除原有培养液，每孔按照CCK8与培养基1：9的体积比加入混合溶液，避光孵育2h后，采用酶标仪于450nm波长处测定各孔吸光度值，并按公式计算出细胞存活率。计算公式为：
细胞存活率＝(A实验组-A空白组)/(A对照组-A空白组)×100％，A为检测波长为450nm出的吸光度。
实验结果如图3所示，10ng/ml～100μg/ml的两种多肽处理24h后，LO2细胞存活率呈现先增加后下降的趋势，表明衍生多肽与LO2细胞存在低浓度促进细胞存活率，高浓度抑制细胞存活率的剂量反应关系。
实施例4—qPCR法检测衍生多肽对LO2细胞端粒长度的影响
实施步骤：将对数生长期细胞随机分为对照组、模型组、PLGTQ和VLNPW组，空白对照组给予完全DMEM完全培养基作为培养液，模型组给予含40μmol/L t-BHP的培养液培养24h，PLGTQ和VLNPW组先分别给予含1μg/mlPLGTQ和0.1μg/mlVLNPW的多肽培养24h，再更换含40μmol/L t-BHP的培养液继续培养24h。分别对各组细胞干预后使用DNA提取试剂盒提取各组细胞总DNA，测定其浓度后以36B4为内参进行PCR扩增检测相对端粒长度，具体方法参照实施例1。
实验结果如图4所示，与模型组相比，PLGTQ和VLNPW组端粒长度显著增加，表明两种多肽可以有效缓解LO2细胞端粒长度的缩短速度。
实施例5—酸奶衍生多肽对细胞衰老模型抗氧化酶和氧化应激产物的影响
实施步骤：按照实施例4设置实验分组和药物处理。细胞处理结束后，按照试剂盒说明书操作，使用酶标仪检测并计算LO2细胞抗氧化酶GSH-Px、CAT和SOD活性以及氧化应激代谢产物ROS水平和MDA水平。
实验结果如图5所示，与模型组相比，PLGTQ组和VLNPW组抗氧化酶GSH-Px、CAT和SOD的活性显著增加，氧化应激产物ROS和MDA水平显著降低，表明两种多肽可以显著抑制LO2细胞内氧化应激产物的聚集并增强细胞抗氧化能力。
实施例6—酸奶衍生多肽对细胞衰老模型凋亡率的影响
实施步骤：按照实施例4设置实验分组和药物处理。细胞处理结束后，用胰酶消化并收集经干预处理后的各组LO2细胞，按照Annexin FITC/PI细胞凋亡检测试剂盒中说明书进行操作，流式细胞仪检测并计算细胞凋亡率，每个样本重复3次。
实验结果如图6所示，对照组细胞凋亡率为2.1％，模型组细胞凋亡率为15.5％，与对照组相比显著增加，PLGTQ和VLNPW组细胞凋亡率分别为5.6％和6.8％，与模型组相比显著降低，表明两种多肽可以有效缓解t-BHP引起的细胞凋亡。
实施例7—酸奶衍生多肽对细胞衰老模型TERT和c-Myc蛋白表达的影响
实施步骤：按照实施例4设置实验分组和药物处理。细胞处理结束后，提取各组细胞总蛋白，使用Bradford法对蛋白进行定量，Western blot法检测TERT和c-Myc蛋白表达水平。
实验结果如图7所示，与模型组相比，PLGTQ和VLNPW组c-Myc蛋白表达水平显著上调，同时TERT蛋白表达显著升高，表明两种多肽可以显著增加端粒酶活性。
综上所述，本发明首次证实了酸奶衍生多肽PLGTQ和VLNPW能够通过调节c-Myc/TERT信号通路和氧化应激水平延缓衰老模型细胞端粒磨损速度。同时，本发明证实了酸奶衍生多肽PLGTQ和VLNPW能够降低衰老模型小鼠脑内β-半乳糖苷酶活性，提高抗氧化能力，延缓端粒磨损速度，从而发挥抗衰老作用。因此，本发明提供了将酸奶衍生多肽PLGTQ和VLNPW在延缓端粒磨损及抗衰老中的应用，为开发和扩大酸奶衍生多肽的抗衰老提供了基础。
本领域的普通技术人员将会意识到，这里所述的实施例是为了帮助读者理解本发明的原理，应被理解为本发明的保护范围并不局限于这样的特别陈述和实施例。本领域的普通技术人员可以根据本发明公开的这些技术启示做出各种不脱离本发明实质的其它各种具体变形和组合，这些变形和组合仍然在本发明的保护范围内。
Claims (6)
1.酸奶衍生多肽在制备延缓端粒缩短药物中的应用。
2.根据权利要求1所述的酸奶衍生多肽在制备延缓端粒缩短药物中的应用，其特征在于，所述酸奶衍生多肽为具有序列PLGTQ或VLNPW的多肽。
3.根据权利要求1或2所述的酸奶衍生多肽在制备延缓端粒缩短药物中的应用，其特征在于，所述药物剂型为片剂、胶囊、颗粒剂、缓释剂或注射剂。
4.酸奶衍生多肽在制备抗衰老药物中的应用。
5.根据权利要求4所述的酸奶衍生多肽在制备抗衰老药物中的应用，其特征在于，所述酸奶衍生多肽为具有序列PLGTQ或VLNPW的多肽。
6.根据权利要求4或5所述的酸奶衍生多肽在制备抗衰老药物中的应用，其特征在于，所述药物剂型为片剂、胶囊、颗粒剂、缓释剂或注射剂。
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211021600.7A CN115252759A (zh) | 2022-08-24 | 2022-08-24 | 酸奶衍生多肽在制备延缓端粒缩短药物及抗衰老药物中的应用 |
| PCT/CN2023/114678 WO2024041604A1 (zh) | 2022-08-24 | 2023-08-24 | 酸奶衍生多肽在制备延缓端粒缩短药物及抗衰老药物中的应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211021600.7A CN115252759A (zh) | 2022-08-24 | 2022-08-24 | 酸奶衍生多肽在制备延缓端粒缩短药物及抗衰老药物中的应用 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115252759A true CN115252759A (zh) | 2022-11-01 |
Family
ID=83752931
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211021600.7A Pending CN115252759A (zh) | 2022-08-24 | 2022-08-24 | 酸奶衍生多肽在制备延缓端粒缩短药物及抗衰老药物中的应用 |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN115252759A (zh) |
| WO (1) | WO2024041604A1 (zh) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2024041604A1 (zh) * | 2022-08-24 | 2024-02-29 | 四川大学华西第二医院 | 酸奶衍生多肽在制备延缓端粒缩短药物及抗衰老药物中的应用 |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107903314A (zh) * | 2017-11-14 | 2018-04-13 | 上海交通大学 | 一种生物活性多肽eviesppeintv及其制备方法和应用 |
| CN108794601A (zh) * | 2018-06-29 | 2018-11-13 | 浙江辉肽生命健康科技有限公司 | 一种生物活性多肽pllpqsl及其制备方法和应用 |
| CN111778256A (zh) * | 2020-04-20 | 2020-10-16 | 广东省微生物研究所（广东省微生物分析检测中心） | CircRNA PVT1及其肽段PVT1-104aa在抗衰老方面的应用 |
| CN113995761A (zh) * | 2021-09-28 | 2022-02-01 | 浙江寿仙谷植物药研究院有限公司 | 灵芝酸a在延缓衰老中的应用 |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2296311A1 (en) * | 1999-01-28 | 2000-07-28 | Universite Laval | Enzymatic hydrolysate of milk proteins |
| WO2006068480A2 (en) * | 2004-12-23 | 2006-06-29 | Campina Nederland Holding B.V. | Protein hydrolysate enriched in peptides inhibiting dpp-iv and their use |
| CN103232528B (zh) * | 2012-12-12 | 2014-12-24 | 上海交通大学 | 一种生物活性多肽delq及其制备和应用 |
| CN106265654A (zh) * | 2016-08-12 | 2017-01-04 | 上海交通大学 | Adjudin在制备延缓衰老药物中的应用 |
| CN110759970A (zh) * | 2018-07-27 | 2020-02-07 | 中国科学院大连化学物理研究所 | 一种源于发酵牛乳的抗氧化多肽及其应用和添加剂 |
| CN115252759A (zh) * | 2022-08-24 | 2022-11-01 | 四川大学华西第二医院 | 酸奶衍生多肽在制备延缓端粒缩短药物及抗衰老药物中的应用 |
-
2022
- 2022-08-24 CN CN202211021600.7A patent/CN115252759A/zh active Pending
-
2023
- 2023-08-24 WO PCT/CN2023/114678 patent/WO2024041604A1/zh unknown
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107903314A (zh) * | 2017-11-14 | 2018-04-13 | 上海交通大学 | 一种生物活性多肽eviesppeintv及其制备方法和应用 |
| CN108794601A (zh) * | 2018-06-29 | 2018-11-13 | 浙江辉肽生命健康科技有限公司 | 一种生物活性多肽pllpqsl及其制备方法和应用 |
| CN111778256A (zh) * | 2020-04-20 | 2020-10-16 | 广东省微生物研究所（广东省微生物分析检测中心） | CircRNA PVT1及其肽段PVT1-104aa在抗衰老方面的应用 |
| CN113995761A (zh) * | 2021-09-28 | 2022-02-01 | 浙江寿仙谷植物药研究院有限公司 | 灵芝酸a在延缓衰老中的应用 |
Non-Patent Citations (1)
| Title |
|---|
| 吕嘉琦等: "酸奶衍生多肽对人肝细胞衰老模型端粒长度的影响及机制研究", 现代预防医学, vol. 48, no. 24, 25 December 2021 (2021-12-25), pages 4497 - 4503 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2024041604A1 (zh) * | 2022-08-24 | 2024-02-29 | 四川大学华西第二医院 | 酸奶衍生多肽在制备延缓端粒缩短药物及抗衰老药物中的应用 |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2024041604A1 (zh) | 2024-02-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP2022063337A (ja) | 微生物共同体の投与により乳生産を改善する方法 | |
| Pampouille et al. | Differential expression and co-expression gene network analyses reveal molecular mechanisms and candidate biomarkers involved in breast muscle myopathies in chicken | |
| Wang et al. | Deubiquitinating enzyme CYLD mediates pressure overload-induced cardiac maladaptive remodeling and dysfunction via downregulating Nrf2 | |
| Wassie et al. | Dietary Enteromorpha polysaccharide enhances intestinal immune response, integrity, and caecal microbial activity of broiler chickens | |
| Liu et al. | In vitro effects of Tartary buckwheat-derived nanovesicles on gut microbiota | |
| Gatta et al. | Gene expression profiles of cumulus cells obtained from women treated with recombinant human luteinizing hormone+ recombinant human follicle-stimulating hormone or highly purified human menopausal gonadotropin versus recombinant human follicle-stimulating hormone alone | |
| Sabino et al. | Oregano dietary supplementation modifies the liver transcriptome profile in broilers: RNASeq analysis | |
| Bonnefont et al. | Genetic susceptibility to S. aureus mastitis in sheep: differential expression of mammary epithelial cells in response to live bacteria or supernatant | |
| WO2024041604A1 (zh) | 酸奶衍生多肽在制备延缓端粒缩短药物及抗衰老药物中的应用 | |
| Reddy et al. | Effect of different early weaning regimens for calves on adipogenic gene expression in Hanwoo loin at the fattening stage | |
| Li et al. | Influence of cecotrophy on fat metabolism mediated by caecal microorganisms in New Zealand white rabbits | |
| Wang et al. | “Dialogue” between Caco-2 and DCs regulated by Ganoderma atrum polysaccharide in intestinal-like Caco-2/DCs co-culture model | |
| Hassan et al. | Effects of probiotic feed additives (biosol and Zemos) on growth and related genes in broiler chickens | |
| Fok et al. | Short-term rapamycin treatment in mice has few effects on the transcriptome of white adipose tissue compared to dietary restriction | |
| Tan et al. | Traditional Chinese medicine Bao Gan Ning increase phosphorylation of CREB in liver fibrosis in vivo and in vitro | |
| Zhao et al. | Dietary supplementation with Dendrobium officinale leaves improves growth, antioxidant status, immune function, and gut health in broilers | |
| Oh et al. | Culturing characteristics of Hanwoo myosatellite cells and C2C12 cells incubated at 37 C and 39 C for cultured meat | |
| CN104017746A (zh) | 乳杆菌、其组合物与治疗自体免疫疾病及并发症的用途 | |
| Liu et al. | Transcriptome sequencing analysis of bovine mammary epithelial cells induced by lipopolysaccharide | |
| Wu et al. | Effect of Clerodendranthus spicatus (Thunb.) CY Wu on the exercise ability of D-galactose-induced oxidative aging mice | |
| Ouyang et al. | Potential mechanism of the PDE-cAMP-related network action on hepatopancreatic necrosis syndrome of Chinese mitten crab (Eriocheir sinensis) | |
| Oommen et al. | Genome wide responses of murine lungs to dietary α-tocopherol | |
| CN112280853B (zh) | 一种肥胖表型分子标志物及其应用 | |
| Wang et al. | 1, 25 (OH) 2D3 alleviates oxidative stress and inflammation through up-regulating HMGCS2 in DSS-induced colitis | |
| Overturf et al. | Microarray analysis of differential utilization of plant-based diets by rainbow trout |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |