WO2024041604A1 - Use of yogurt-derived polypeptide in preparation of drug for delaying telomere shortening and anti-aging drug - Google Patents
Use of yogurt-derived polypeptide in preparation of drug for delaying telomere shortening and anti-aging drug Download PDFInfo
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Classifications
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- 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
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- 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
Definitions
- the invention belongs to the technical fields of biotechnology and biopharmaceuticals, and relates to the application of yogurt-derived polypeptides in the preparation of drugs that delay telomere shortening and anti-aging drugs.
- Telomeres DNA-protein complexes that exist at the ends of linear chromosomes in eukaryotic cells. Together with telomere-binding proteins, they form the special "cap" structure of the chromosome. Its main function is to maintain the integrity of the chromosome and protect it from damage. . As cells divide, telomeres will progressively shorten. When telomeres shorten to a certain threshold, cells will send out damage signals and initiate cell senescence and apoptosis programs, causing cells to move toward irreversible aging and death. Therefore, the maintenance of telomere length is crucial for cellular aging and even individual aging.
- telomere theory shows that oxidative stress can aggravate telomere wear: on the one hand, guanine-rich telomeric DNA sequences are easily targets of acute oxidative damage; on the other hand, oxidative stress products such as reactive oxygen species Telomere wear can be accelerated by disrupting the telomere length regulation mechanism. Therefore, accumulation of intracellular oxidative damage may be an important cause of telomere attrition.
- telomerase reverse transcriptase activity is another important regulator of telomere length. It uses its own RNA template function and reverse transcriptase activity to synthesize new telomeric DNA repeat sequences, thereby participating in the process of cell proliferation. Maintenance of length.
- Yogurt is made from milk through microbial fermentation.
- the fermentation process not only provides the yogurt with various microbial active metabolites, but also introduces a variety of active peptides with preventive and health care functions. These active peptides are beneficial to the cardiovascular system, nervous system, and intestinal health. Provides multiple health benefits in areas such as and immune defense.
- the active peptides in yogurt can further release small molecule-derived peptides with higher biological activity during the gastrointestinal digestion stage.
- it is unclear whether the active peptides in yogurt can slow down the wear and tear of telomere length through antioxidant effects.
- the purpose of the present invention is to provide the application of yogurt-derived polypeptides in the preparation of telomere-delaying drugs.
- yogurt can slow down the wear and tear of telomere length in aging model mice by enhancing antioxidant capacity.
- low molecular weight derived peptides with potential antioxidant biological activity unique to yogurt were screened out.
- Their sequences are PLGTQ (proline-leucine-glycine-threonine- Glutamine) and VLNPW (Valine-Leucine-Asparagine-Proline-Tryptophan), for the specific screening process, please refer to (Peptide profiling and the bioactivity character of yogurt in the simulated gastrointestinal digestion, Journal of Proteomics , Volume 141, 1 June 2016, Pages 24-46).
- nucleic acid sequences encoding PLGTQ and VLNPW are as follows:
- the present invention provides the use of yogurt-derived polypeptides in preparing drugs for delaying telomere shortening.
- the yogurt-derived polypeptide is a polypeptide having the sequence PLGTQ or VLNPW.
- the application of the yogurt-derived polypeptide provided by the present invention in the preparation of drugs for delaying telomere shortening has no special restrictions on the drug dosage form and preparation method. It can be made into tablets, capsules, granules, and sustained-release agents using conventional preparation methods in this field. , injections and other various dosage forms.
- the present invention further provides the application of the above-mentioned yogurt-derived polypeptide in the preparation of anti-aging drugs.
- the application of the yogurt-derived polypeptide provided by the present invention in the preparation of anti-aging drugs has no special restrictions on the pharmaceutical dosage form and preparation method. It can be made into tablets, capsules, granules, sustained-release agents, and injections using conventional preparation methods in this field. Various dosage forms.
- the application of the yogurt-derived polypeptide provided by the present invention in the preparation of drugs that delay telomere shortening and anti-aging drugs has the following beneficial effects:
- the present invention explores new medical uses of yogurt-derived polypeptides, opens up a new application field, and is expected to become an effective drug for delaying telomere shortening and anti-aging. It also provides a basis for the development and expansion of yogurt-derived polypeptides for anti-aging. .
- Yogurt-derived peptides can slow down the rate of cellular telomere wear by regulating the c-Myc/TERT signaling pathway and oxidative stress levels.
- Yoghurt-derived peptides (such as peptides with the sequences PLGTQ and VLNPW) can reduce ⁇ -galactosidase activity, improve antioxidant capacity, and then delay the rate of telomere wear, thus exerting anti-aging effects.
- Figure 1 shows the test results of telomere length of peripheral blood leukocytes of aging mice under different conditions.
- Figure 2 shows the test results of ⁇ -gal activity in the liver of aging mice under different conditions.
- Figure 3 shows the test results of the CCK8 method for detecting the cell viability of yogurt-derived peptides in the cell senescence model.
- Figure 4 shows the test results of relative telomere length of cell senescence models under different conditions.
- Figure 5 shows the effects of antioxidant enzymes (GSH-Px, CAT and SOD activities) and oxidative stress metabolites (ROS levels and MDA levels) in the cell aging model under different conditions.
- Figure 6 shows the test results of apoptosis rate of cell senescence model under different conditions.
- Figure 7 shows the test results of TERT and c-Myc protein expression in the cell senescence model under different conditions; ⁇ -actin (actin) is used as the internal control.
- Yogurt-derived peptides with the sequences PLGTQ and VLNPW were obtained by conventional technical means, see (Peptide profiling and the bioactivity character of yogurt in the simulatedgastrointestinal digestion, Journal of Proteomics, Volume 141, 1 June 2016, Pages 24-46).
- Example 1 - qPCR method to detect the effect of yogurt-derived polypeptides on telomere length in mice
- Modeling treatment Animals were raised in a well-ventilated environment with a temperature of 20-25°C, a relative humidity of 65-70%, a light-dark alternation cycle of 12 hours. After one week of adaptive feeding, an aging mouse model was prepared by subcutaneously injecting 80 mg/kg D-gal for 6 weeks. At the end of the 6th week, the mice showed slow response, reduced activity, dull hair, and listlessness, indicating aging. The model was built successfully.
- the positive control group vitamin E 100mg/kg/day
- PLGTQ and VLNPW (20mg/kg/day) will be given corresponding doses of drugs by intragastric administration every day for 8 weeks, and the control group will be given sterile saline by intragastric administration. (20mg/kg/day).
- telomere length was detected using 36B4 as the internal reference.
- the cycling conditions were 95°C for 2 min and 95°C for pre-denaturation. Denaturation for 5 s, annealing and extension at 60°C for 10 s, 40 cycles were performed.
- the relative expression of the gene was calculated using the 2 - ⁇ Ct method.
- the primer sequences are shown in Table 1.
- Example 2 Staining method to detect ⁇ -galactosidase activity in mouse liver tissue
- Example 3 CCK8 method to detect the effect of yogurt-derived polypeptides on cell viability in cell senescence model
- LO2 cells were purchased from the Cell Bank of the Shanghai Chinese Academy of Sciences.
- Implementation steps Inoculate LO2 cells in the logarithmic phase into a 96-well plate with a cell density of 5 ⁇ 10 3 cells/well. Each group is set with 6 replicate wells. After the cells adhere to the wall, the corresponding cells are given according to the requirements of different experimental groups. Intervention treatment, the blank group was DMEM complete medium without any cells; the control group was cell culture medium without polypeptides (DMEM complete medium as the culture medium); the experimental group was added with 0.01, 0.05, 0.1, 0.5, 1 respectively. , 5, 10, 50 and 100 ⁇ g/ml polypeptide cell culture medium.
- Cell survival rate (A experimental group - A blank group ) / (A control group - A blank group ) ⁇ 100%, A is the absorbance at the detection wavelength of 450 nm.
- Example 4 - qPCR method to detect the effect of derived polypeptides on telomere length of LO2 cells
- Cells in logarithmic growth phase were randomly divided into control group, model group, PLGTQ and VLNPW groups.
- the blank control group was given complete DMEM medium as culture medium, and the model group was given culture medium containing 40 ⁇ mol/L t-BHP.
- the PLGTQ and VLNPW groups were first given peptides containing 1 ⁇ g/ml PLGTQ and 0.1 ⁇ g/ml VLNPW respectively for 24 hours, and then the culture medium containing 40 ⁇ mol/L t-BHP was replaced and cultured for 24 hours.
- the extraction kit extracted the total DNA of cells in each group, measured its concentration, and then used 36B4 as an internal reference to perform PCR amplification to detect the relative telomere length. For specific methods, refer to Example 1.
- Example 5 Effects of yogurt-derived polypeptides on antioxidant enzymes and oxidative stress products in cellular aging model
- Example 6 Effects of yogurt-derived polypeptides on the apoptosis rate of cell senescence model
- the experimental results are shown in Figure 6.
- the cell apoptosis rate in the control group was 2.1%, and the cell apoptosis rate in the model group was 15.5%, which was significantly increased compared with the control group.
- the cell apoptosis rates in the PLGTQ and VLNPW groups were 5.6% and 6.8 respectively. %, significantly reduced compared with the model group, indicating that the two polypeptides can effectively alleviate cell apoptosis caused by t-BHP.
- Example 7 Effects of yogurt-derived polypeptides on expression of TERT and c-Myc proteins in cellular senescence model
- the present invention has confirmed for the first time that yogurt-derived peptides PLGTQ and VLNPW can slow down the telomere wear rate of aging model cells by regulating the c-Myc/TERT signaling pathway and oxidative stress levels.
- the present invention confirms that the yogurt-derived polypeptides PLGTQ and VLNPW can reduce ⁇ -galactosidase activity in the brains of aging model mice, improve antioxidant capacity, delay telomere wear, and thereby exert anti-aging effects. Therefore, the present invention provides the application of yogurt-derived polypeptides PLGTQ and VLNPW in delaying telomere wear and anti-aging, and provides a basis for developing and expanding the anti-aging of yogurt-derived polypeptides.
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Abstract
The present invention belongs to the fields of biotechnology and biopharmaceutical technology. Disclosed is use of a yogurt-derived polypeptide in the preparation of a drug for delaying telomere shortening and an anti-aging drug. The present invention explores new medical use of the yogurt-derived polypeptide, opens up a new application field, is expected to become an effective drug for delaying telomere shortening and resisting aging, and meanwhile, provides a basis for developing and expanding the anti-aging of the yogurt-derived polypeptide.
Description
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。The invention belongs to the technical fields of biotechnology and biopharmaceuticals, and relates to the application of yogurt-derived polypeptides in the preparation of drugs that delay telomere shortening and anti-aging drugs.
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。Telomeres are DNA-protein complexes that exist at the ends of linear chromosomes in eukaryotic cells. Together with telomere-binding proteins, they form the special "cap" structure of the chromosome. Its main function is to maintain the integrity of the chromosome and protect it from damage. . As cells divide, telomeres will progressively shorten. When telomeres shorten to a certain threshold, cells will send out damage signals and initiate cell senescence and apoptosis programs, causing cells to move toward irreversible aging and death. Therefore, the maintenance of telomere length is crucial for cellular aging and even individual aging. The classic "telomere theory" shows that oxidative stress can aggravate telomere wear: on the one hand, guanine-rich telomeric DNA sequences are easily targets of acute oxidative damage; on the other hand, oxidative stress products such as reactive oxygen species Telomere wear can be accelerated by disrupting the telomere length regulation mechanism. Therefore, accumulation of intracellular oxidative damage may be an important cause of telomere attrition. In addition, telomerase reverse transcriptase activity is another important regulator of telomere length. It uses its own RNA template function and reverse transcriptase activity to synthesize new telomeric DNA repeat sequences, thereby participating in the process of cell proliferation. Maintenance of length.
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。Yogurt is made from milk through microbial fermentation. The fermentation process not only provides the yogurt with various microbial active metabolites, but also introduces a variety of active peptides with preventive and health care functions. These active peptides are beneficial to the cardiovascular system, nervous system, and intestinal health. Provides multiple health benefits in areas such as and immune defense. In addition, the active peptides in yogurt can further release small molecule-derived peptides with higher biological activity during the gastrointestinal digestion stage. However, it is unclear whether the active peptides in yogurt can slow down the wear and tear of telomere length through antioxidant effects.
发明内容Contents of the invention
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。In view of the technical problem that oxidative stress accelerates telomere shortening and leads to irreversible cell aging, the purpose of the present invention is to provide the application of yogurt-derived polypeptides in the preparation of telomere-delaying drugs.
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(Peptide profiling and the bioactivity character of yogurt in the simulated gastrointestinal digestion,Journal of Proteomics,Volume 141,1 June 2016,Pages 24-46)。Studies have found that yogurt can slow down the wear and tear of telomere length in aging model mice by enhancing antioxidant capacity. By comparing with the polypeptide types after digestion of milk, low molecular weight derived peptides with potential antioxidant biological activity unique to yogurt were screened out. Their sequences are PLGTQ (proline-leucine-glycine-threonine- Glutamine) and VLNPW (Valine-Leucine-Asparagine-Proline-Tryptophan), for the specific screening process, please refer to (Peptide profiling and the bioactivity character of yogurt in the simulated gastrointestinal digestion, Journal of Proteomics , Volume 141, 1 June 2016, Pages 24-46).
编码PLGTQ和VLNPW的核酸序列如下所示：
The nucleic acid sequences encoding PLGTQ and VLNPW are as follows:
PLGTQ:ccgctgggcacccagPLGTQ:ccgctgggcacccag
VLNPW:gtgctgaacccgtggVLNPW: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。The present invention provides the use of yogurt-derived polypeptides in preparing drugs for delaying telomere shortening. The yogurt-derived polypeptide is a polypeptide having the sequence PLGTQ or VLNPW. The application of the yogurt-derived polypeptide provided by the present invention in the preparation of drugs for delaying telomere shortening has no special restrictions on the drug dosage form and preparation method. It can be made into tablets, capsules, granules, and sustained-release agents using conventional preparation methods in this field. , injections and other various dosage forms.
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。In view that the above-mentioned yogurt-derived polypeptide can effectively delay telomere shortening, thereby delaying aging and death, the present invention further provides the application of the above-mentioned yogurt-derived polypeptide in the preparation of anti-aging drugs. The application of the yogurt-derived polypeptide provided by the present invention in the preparation of anti-aging drugs has no special restrictions on the pharmaceutical dosage form and preparation method. It can be made into tablets, capsules, granules, sustained-release agents, and injections using conventional preparation methods in this field. Various dosage forms.
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：Compared with the existing technology, the application of the yogurt-derived polypeptide provided by the present invention in the preparation of drugs that delay telomere shortening and anti-aging drugs has the following beneficial effects:
(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。(1) The present invention explores new medical uses of yogurt-derived polypeptides, opens up a new application field, and is expected to become an effective drug for delaying telomere shortening and anti-aging. It also provides a basis for the development and expansion of yogurt-derived polypeptides for anti-aging. .
(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。(2) Yogurt-derived peptides (such as peptides with the sequences PLGTQ and VLNPW) can slow down the rate of cellular telomere wear by regulating the c-Myc/TERT signaling pathway and oxidative stress levels.
(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。(3) Yoghurt-derived peptides (such as peptides with the sequences PLGTQ and VLNPW) can reduce β-galactosidase activity, improve antioxidant capacity, and then delay the rate of telomere wear, thus exerting anti-aging effects.
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。In order to explain the embodiments of the present invention or solutions in the prior art more clearly, the following will briefly introduce the drawings needed to describe the embodiments or the prior art.
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。Figure 1 shows the test results of telomere length of peripheral blood leukocytes of aging mice under different conditions.
图2为不同条件下衰老小鼠肝脏β-gal活性测试结果。Figure 2 shows the test results of β-gal activity in the liver of aging mice under different conditions.
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。Figure 3 shows the test results of the CCK8 method for detecting the cell viability of yogurt-derived peptides in the cell senescence model.
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。Figure 4 shows the test results of relative telomere length of cell senescence models under different conditions.
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。Figure 5 shows the effects of antioxidant enzymes (GSH-Px, CAT and SOD activities) and oxidative stress metabolites (ROS levels and MDA levels) in the cell aging model under different conditions.
图6为不同条件下细胞衰老模型凋亡率的测试结果。Figure 6 shows the test results of apoptosis rate of cell senescence model under different conditions.
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。Figure 7 shows the test results of TERT and c-Myc protein expression in the cell senescence model under different conditions; β-actin (actin) is used as the internal control.
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。The technical solutions of various embodiments of the present invention will be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without any creative efforts belong to the present invention.
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(Peptide profiling and the bioactivity character of yogurt in the simulatedgastrointestinal digestion,Journal of Proteomics,Volume 141,1 June 2016,Pages 24-46)。Yogurt-derived peptides with the sequences PLGTQ and VLNPW were obtained by conventional technical means, see (Peptide profiling and the bioactivity character of yogurt in the simulatedgastrointestinal digestion, Journal of Proteomics, Volume 141, 1 June 2016, 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Example 1 - qPCR method to detect the effect of yogurt-derived polypeptides on telomere length in mice
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。Experimental materials: Kunming mice (SPF grade), male, 3 months old, purchased from Beijing Vitong Lever Experimental Animal Company.
实施步骤：Implementation steps:
(1)动物饲养及分组：(1) Animal feeding and grouping:
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。80 5-week-old male Kunming mice were randomly divided into blank control group, aging model group, positive control group, PLGTQ and VLNPW. Except for the blank control group, the other four groups were all subjected to modeling treatment.
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。Modeling treatment: Animals were raised in a well-ventilated environment with a temperature of 20-25°C, a relative humidity of 65-70%, a light-dark alternation cycle of 12 hours. After one week of adaptive feeding, an aging mouse model was prepared by subcutaneously injecting 80 mg/kg D-gal for 6 weeks. At the end of the 6th week, the mice showed slow response, reduced activity, dull hair, and listlessness, indicating aging. The model was built successfully.
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)。After the model is successfully constructed, the positive control group (vitamin E 100mg/kg/day), PLGTQ and VLNPW (20mg/kg/day) will be given corresponding doses of drugs by intragastric administration every day for 8 weeks, and the control group will be given sterile saline by intragastric administration. (20mg/kg/day).
(2)端粒长度检测：(2) Telomere length detection:
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2-△△Ct法计算基因相对表达量，引物序列见表1。After extracting DNA from peripheral blood leukocytes of mice in each group according to the instructions of the DNA extraction kit, PCR amplification was performed to detect the relative telomere length using 36B4 as the internal reference. Each sample was tested three times. The cycling conditions were 95°C for 2 min and 95°C for pre-denaturation. Denaturation for 5 s, annealing and extension at 60°C for 10 s, 40 cycles were performed. The relative expression of the gene was calculated using the 2 -ΔΔCt method. The primer sequences are shown in Table 1.
表1 引物名称和序列
Table 1 Primer names and sequences
Table 1 Primer names and sequences
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。The experimental results are shown in Figure 1. Compared with the model group, the telomere length of the positive control group, PLGTQ and VLNPW groups was significantly extended, and the differences were statistically significant, indicating that the two polypeptides can have the same significant effect as the anti-aging positive control vitamin E. Alleviating the wear rate of telomere length in peripheral blood of aging model mice.
实施例2—染色法检测小鼠肝组织β-半乳糖酶活性Example 2 - Staining method to detect β-galactosidase activity in mouse liver tissue
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。Implementation steps: Set up animal groups according to Example 1 and perform drug treatment. The liver tissues of mice in each group after drug intervention were dissected, fixed with paraformaldehyde for 24 hours, soaked in sucrose overnight, and then frozen sections were prepared. They were developed with incubation solution overnight, counterstained with neutral red, and observed under an oil microscope in each group. Number of blue positive signal particles.
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。The experimental results are shown in Figure 2. Compared with the model group, the number of blue signal particles in the positive control group, PLGTQ and VLNPW groups was significantly reduced, indicating that β-gal activity was weakened, suggesting that the two polypeptides have the same remission model as vitamin E. Effects of aging in mice.
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Example 3 - CCK8 method to detect the effect of yogurt-derived polypeptides on cell viability in cell senescence model
实验材料：LO2细胞购于上海中科院典藏细胞库Experimental materials: LO2 cells were purchased from the Cell Bank of the Shanghai Chinese Academy of Sciences.
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。Implementation steps: Inoculate LO2 cells in the logarithmic phase into a 96-well plate with a cell density of 5×10 3 cells/well. Each group is set with 6 replicate wells. After the cells adhere to the wall, the corresponding cells are given according to the requirements of different experimental groups. Intervention treatment, the blank group was DMEM complete medium without any cells; the control group was cell culture medium without polypeptides (DMEM complete medium as the culture medium); the experimental group was added with 0.01, 0.05, 0.1, 0.5, 1 respectively. , 5, 10, 50 and 100μg/ml polypeptide cell culture medium.
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：After 24 hours of intervention, the original culture medium was removed, and the mixed solution was added to each well according to the volume ratio of CCK8 to medium 1:9. After incubation in the dark for 2 hours, the absorbance value of each well was measured at a wavelength of 450 nm using a microplate reader, and according to the formula Cell viability was calculated. The calculation formula is:
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。Cell survival rate = (A experimental group - A blank group ) / (A control group - A blank group ) × 100%, A is the absorbance at the detection wavelength of 450 nm.
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。The experimental results are shown in Figure 3. After being treated with two polypeptides of 10ng/ml~100μg/ml for 24 hours, the survival rate of LO2 cells showed a trend of first increasing and then decreasing, indicating that the presence of low concentrations of derived polypeptides and LO2 cells promotes cell survival rate and high Dose-response relationship for concentration inhibition of cell viability.
实施例4—qPCR法检测衍生多肽对LO2细胞端粒长度的影响Example 4 - qPCR method to detect the effect of derived polypeptides on telomere length of LO2 cells
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。Implementation steps: Cells in logarithmic growth phase were randomly divided into control group, model group, PLGTQ and VLNPW groups. The blank control group was given complete DMEM medium as culture medium, and the model group was given culture medium containing 40 μmol/L t-BHP. At 24 hours, the PLGTQ and VLNPW groups were first given peptides containing 1 μg/ml PLGTQ and 0.1 μg/ml VLNPW respectively for 24 hours, and then the culture medium containing 40 μmol/L t-BHP was replaced and cultured for 24 hours. After interfering with cells in each group, DNA was used The extraction kit extracted the total DNA of cells in each group, measured its concentration, and then used 36B4 as an internal reference to perform PCR amplification to detect the relative telomere length. For specific methods, refer to Example 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。The experimental results are shown in Figure 4. Compared with the model group, the telomere length of the PLGTQ and VLNPW groups increased significantly, indicating that the two polypeptides can effectively alleviate the shortening of telomere length in LO2 cells.
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Example 5—Effects of yogurt-derived polypeptides on antioxidant enzymes and oxidative stress products in cellular aging model
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。Implementation steps: Set the experimental grouping and drug treatment according to Example 4. After cell treatment, follow the instructions of the kit and use a microplate reader to detect and calculate the activities of antioxidant enzymes GSH-Px, CAT and SOD as well as the levels of oxidative stress metabolites ROS and MDA in LO2 cells.
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。The experimental results are shown in Figure 5. Compared with the model group, the activities of antioxidant enzymes GSH-Px, CAT and SOD in the PLGTQ group and VLNPW group were significantly increased, and the levels of oxidative stress products ROS and MDA were significantly reduced, indicating that the two polypeptides can Significantly inhibits the accumulation of oxidative stress products in LO2 cells and enhances cellular antioxidant capacity.
实施例6—酸奶衍生多肽对细胞衰老模型凋亡率的影响Example 6—Effects of yogurt-derived polypeptides on the apoptosis rate of cell senescence model
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。Implementation steps: Set the experimental grouping and drug treatment according to Example 4. After the cell treatment, use trypsin to digest and collect the LO2 cells in each group after intervention. Follow the instructions in the Annexin FITC/PI apoptosis detection kit. The cell apoptosis rate is detected and calculated with a flow cytometer. Each sample was repeated 3 times.
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。The experimental results are shown in Figure 6. The cell apoptosis rate in the control group was 2.1%, and the cell apoptosis rate in the model group was 15.5%, which was significantly increased compared with the control group. The cell apoptosis rates in the PLGTQ and VLNPW groups were 5.6% and 6.8 respectively. %, significantly reduced compared with the model group, indicating that the two polypeptides can effectively alleviate cell apoptosis caused by t-BHP.
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Example 7—Effects of yogurt-derived polypeptides on expression of TERT and c-Myc proteins in cellular senescence model
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。Implementation steps: Set the experimental grouping and drug treatment according to Example 4. After the cell treatment, the total protein of cells in each group was extracted, the protein was quantified using the Bradford method, and the expression levels of TERT and c-Myc proteins were detected by Western blot method.
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。The experimental results are shown in Figure 7. Compared with the model group, the c-Myc protein expression level in the PLGTQ and VLNPW groups was significantly increased, and the TERT protein expression was significantly increased, indicating that the two polypeptides can significantly increase telomerase activity.
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。In summary, the present invention has confirmed for the first time that yogurt-derived peptides PLGTQ and VLNPW can slow down the telomere wear rate of aging model cells by regulating the c-Myc/TERT signaling pathway and oxidative stress levels. At the same time, the present invention confirms that the yogurt-derived polypeptides PLGTQ and VLNPW can reduce β-galactosidase activity in the brains of aging model mice, improve antioxidant capacity, delay telomere wear, and thereby exert anti-aging effects. Therefore, the present invention provides the application of yogurt-derived polypeptides PLGTQ and VLNPW in delaying telomere wear and anti-aging, and provides a basis for developing and expanding the anti-aging of yogurt-derived polypeptides.
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。
Those of ordinary skill in the art will appreciate that the embodiments described herein are provided to assist the reader in understanding the present invention. Principle, it should be understood that the scope of the present invention is not limited to such specific statements and examples. Those of ordinary skill in the art can make various other specific modifications and combinations based on the technical teachings disclosed in the present invention without departing from the essence of the present invention, and these modifications and combinations are still within the protection scope of the present invention.
Claims (6)
- 酸奶衍生多肽在制备延缓端粒缩短药物中的应用。Application of yogurt-derived peptides in the preparation of drugs that delay telomere shortening.
- 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。The application of yogurt-derived polypeptide in the preparation of drugs for delaying telomere shortening according to claim 1, characterized in that the yogurt-derived polypeptide is a polypeptide with the sequence PLGTQ or VLNPW.
- 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。The application of the yogurt-derived polypeptide in the preparation of drugs for delaying telomere shortening according to claim 1 or 2, characterized in that the pharmaceutical dosage form is a tablet, capsule, granule, sustained release agent or injection.
- 酸奶衍生多肽在制备抗衰老药物中的应用。Application of yogurt-derived peptides in the preparation of anti-aging drugs.
- 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。The application of yogurt-derived polypeptide in the preparation of anti-aging drugs according to claim 4, characterized in that the yogurt-derived polypeptide is a polypeptide with the sequence PLGTQ or VLNPW.
- 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。 The application of the yogurt-derived polypeptide in the preparation of anti-aging drugs according to claim 4 or 5, characterized in that the pharmaceutical dosage form is a tablet, capsule, granule, sustained release agent or injection.
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