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WO2023202099A1 - 甘油磷酸胆碱在调控细胞nad+水平和分布中的应用 - Google Patents

甘油磷酸胆碱在调控细胞nad+水平和分布中的应用 Download PDF

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WO2023202099A1
WO2023202099A1 PCT/CN2022/138175 CN2022138175W WO2023202099A1 WO 2023202099 A1 WO2023202099 A1 WO 2023202099A1 CN 2022138175 W CN2022138175 W CN 2022138175W WO 2023202099 A1 WO2023202099 A1 WO 2023202099A1
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adenine dinucleotide
nicotinamide adenine
glycerophosphocholine
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PCT/CN2022/138175
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French (fr)
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於邱黎阳
陈柳青
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深圳先进技术研究院
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/661—Phosphorus acids or esters thereof not having P—C bonds, e.g. fosfosal, dichlorvos, malathion or mevinphos
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/683—Diesters of a phosphorus acid with two hydroxy compounds, e.g. phosphatidylinositols
    • A61K31/685—Diesters of a phosphorus acid with two hydroxy compounds, e.g. phosphatidylinositols one of the hydroxy compounds having nitrogen atoms, e.g. phosphatidylserine, lecithin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7084—Compounds having two nucleosides or nucleotides, e.g. nicotinamide-adenine dinucleotide, flavine-adenine dinucleotide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/06—Phosphorus compounds without P—C bonds
    • C07F9/08—Esters of oxyacids of phosphorus
    • C07F9/09—Esters of phosphoric acids
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the invention belongs to the field of biotechnology and relates to the application of a compound. Specifically, it is the application of glycerophosphocholine in regulating the level and distribution of nicotinamide adenine dinucleotide (NAD + ) in cells.
  • NAD + nicotinamide adenine dinucleotide
  • Nicotinamide adenine dinucleotide has important functions for all organisms: it participates in hundreds of redox reactions as a coenzyme factor; it is a substrate for dozens of regulatory enzyme molecules such as PARPs and SIRTs; Involved in atypical RNA-capping modifications. Therefore, intracellular NAD + has an important impact on cell metabolism, cell signaling, and gene expression regulation. Intracellular NAD + levels undergo dynamic changes in numerous physiological phenomena such as aging, neurodegeneration, kidney injury, obesity, and diabetes. In eukaryotic cells, the synthesis and consumption of NAD + are highly compartmentalized and widely distributed in the cytoplasm, nucleus, mitochondria, endoplasmic reticulum, and Golgi apparatus.
  • lipogenesis signals can rapidly induce the expression of cytoplasmic NAD + synthase NMNAT-2 and compete with the nuclear NAD + synthase NMNAT-1 for the common substrate nicotinamide mononucleotide, resulting in a sharp decrease in nuclear NAD + levels and
  • the original NAD + spatiotemporal distribution is imbalanced, and further induces adipogenic transcription of cells and induces adipocyte differentiation, while reversing the decrease in nuclear NAD + can reduce adipogenic transcription of cells.
  • the imbalance process of the spatiotemporal distribution of organelle NAD + plays a significant role in the regulation of adipocyte differentiation and neuroblastoma function. Reshaping the spatiotemporal distribution of organelle NAD + is expected to play a positive role in regulating adipogenic differentiation and neuroblastoma function.
  • cytoplasmic NAD + plays an important role in ovarian cancer by regulating translation and maintaining protein homeostasis.
  • the expression of cytoplasmic NMNAT-2 is highly upregulated in ovarian cancer and competes with nuclear NAD + synthesis, resulting in a decrease in nuclear NAD + levels and an imbalance in the spatiotemporal distribution of NAD + .
  • NMNAT-2 can maintain the catalytic activity of the mono(ADP-ribosyl)transferase PARP-16, and PARP-16 can mono-ADP-ribosylate ribosomal proteins.
  • Downregulation of NMNAT-2 or PARP-16 results in inhibition of mono-ADP-ribosylation, increased polyribosome binding, enhanced translation of specific mRNAs, aggregation of related protein products, and reduced ovarian cancer cell growth.
  • the distribution of NAD + in mitochondria and nuclei regulates many disease-related processes the development of NAD + organelle-specific regulatory molecules is of great significance and is a promising metabolic drug and nutritional intervention strategy.
  • L- ⁇ -glycerophosphoryl choline (Alpha-GPC for short) is composed of choline, glycerol and phosphate, and its structure is shown in Figure 1.
  • Alpha-GPC naturally exists in the human body and is the precursor for the synthesis of the neurotransmitter acetylcholine. It has important nutritional and health functions and medicinal value. Numerous academic studies and clinical trials have shown that Alpha-GPC has the effects of improving cognitive function, anti-hyperlipidemia and protecting blood vessels, and can be used to treat diseases such as Alzheimer's disease and schizophrenia. However, there are currently no research reports on the relationship between Alpha-GPC and NAD + .
  • the present invention aims to provide the application of glycerophosphocholine in regulating the level and distribution of cellular nicotinamide adenine dinucleotide.
  • a first aspect of the present invention provides the use of glycerophosphocholine in regulating cellular nicotinamide adenine dinucleotide levels and distribution ratios.
  • the glycerophosphocholine is used to regulate the level and distribution of nicotinamide adenine dinucleotide in subcellular structures of living cells.
  • subcellular structure is the nucleus or mitochondria.
  • the glycerophosphocholine is used to increase the level and distribution of nicotinamide adenine dinucleotide in the nucleus, and/or reduce the level and distribution of nicotinamide adenine dinucleotide in the mitochondria.
  • a second aspect of the present invention provides the use of glycerophosphocholine in the preparation of pharmaceutical compositions for preventing and/or treating diseases related to the regulation of cellular nicotinamide adenine dinucleotide metabolism.
  • the glycerophosphocholine is used to prepare a pharmaceutical composition for preventing and/or treating diseases related to the metabolism regulation of nicotinamide adenine dinucleotide in subcellular structures.
  • the glycerophosphocholine is used to prepare diseases related to the prevention and/or treatment of reduced levels and distribution of nicotinamide adenine dinucleotide in the nucleus, and/or increased levels and distribution of mitochondrial nicotinamide adenine dinucleotide.
  • pharmaceutical composition comprising
  • the diseases related to the regulation of cellular nicotinamide adenine dinucleotide metabolism include abnormal lipid cell differentiation, mitochondrial energy metabolism diseases, Alzheimer's disease manifested by subcellular NAD + distribution imbalance, and pseudohypoxia. wait.
  • the third aspect of the present invention provides glycerophosphocholine in the preparation of health products and dietary supplements for regulating the level and distribution of nicotinamide adenine dinucleotide in cells, anti-aging, extending cell life, and assisting in lowering blood lipids or lowering blood sugar.
  • a fourth aspect of the present invention provides a pharmaceutical preparation for regulating the level and distribution of nicotinamide adenine dinucleotide in cells, the active ingredient of the pharmaceutical preparation includes glycerophosphocholine;
  • the pharmaceutical preparation is used to increase nuclear nicotinamide adenine dinucleotide levels and distribution, and/or to decrease mitochondrial nicotinamide adenine dinucleotide levels and distribution.
  • a fifth aspect of the present invention provides a pharmaceutical composition for preventing and/or treating diseases related to cellular nicotinamide adenine dinucleotide metabolism regulation, the active ingredient of the pharmaceutical composition includes glycerophosphocholine;
  • the pharmaceutical composition is used to prevent and/or treat diseases related to a decrease in the level and distribution of nicotinamide adenine dinucleotide in the nucleus, and/or an increase in the level and distribution of nicotinamide adenine dinucleotide in the mitochondria.
  • a sixth aspect of the present invention provides a health product, dietary supplement, functional food, medical food or nutritional supplement, the ingredients of which include glycerol phosphate choline;
  • the health products, dietary supplements, functional foods, medical foods or nutritional supplements are used to regulate cellular nicotinamide adenine dinucleotide levels and distribution, anti-aging, extend cell life, assist in lowering blood lipids or assist in Hypoglycemic.
  • Alpha-GPC can specifically regulate the level and distribution of nuclear and mitochondrial NAD + .
  • Alpha-GPC can specifically increase the level of nuclear NAD + and reduce the level of mitochondrial NAD + , which is a disease treatment strategy based on subcellular NAD + metabolic regulation.
  • Alpha-GPC will not cause any acute, subacute or chronic harm to the human body and can be developed to regulate cellular nicotinamide adenine dinucleotide levels and distribution, anti-aging, extend cell life, assist in lowering blood lipids or assist in lowering blood sugar. of health products, dietary supplements, functional foods, medical foods or nutritional supplements.
  • Figure 1 is a schematic structural diagram of compound Alpha-GPC.
  • Figure 2(A) is a schematic diagram of the working of probes NADS2.0 and NADS3.0.
  • Figure 3 shows the ratio of luminescence intensity at 515 nm and 440 nm in HEK 293T cells that express the mitochondria-specific probe NADS2.0 after being treated with a combination of Alpha-GPC and FK866 at different concentrations for 24 hours.
  • B Ratio of luminescence intensity at 515 nm and 440 nm in HEK 293T cells that specifically express the probe NADS2.0 after being treated with a combination of Alpha-GPC and FK866 at different concentrations for 24 hours. All P values are using unpaired two-tailed Student’s t tests calculated: *p ⁇ 0.05, **p ⁇ 0.01; ***p ⁇ 0.001; ****p ⁇ 0.0001.
  • Figure 4(A) is a micrograph of the FRET signal of HEK 293T cells stably transfected with mitochondria- or nucleus-specific expression probe NADS3.0 after treatment with Alpha-GPC.
  • B Statistical histogram of FRET (RFP/GFP) signals in HEK 293T cells stably transfected with mitochondria- or nucleus-specific expression probe NADS3.0 after treatment with Alpha-GPC.
  • Example 1 Preparation of HEK 293T cells stably transduced with NAD + molecular probe
  • NAD + molecular probes used were the probe NADS2.0 based on bioluminescence resonance energy transfer and the probe NADS3.0 based on fluorescence resonance energy transfer.
  • a nuclear localization peptide sequence NPKKKRKV
  • MTS mitochondrial localization sequence
  • Nuc-NADS2.0 SEQ ID NO. 1
  • Nuc-NADS3.0 SEQ ID NO. 1
  • Mito-NADS2.0 (SEQ ID NO. 3), and Mito-NADS3.0 (SEQ ID NO. 4) realize nuclear and mitochondrial specificity of NAD + molecular probes sexual expression.
  • the coding genes of these four probes (Nuc-NADS2.0, Nuc-NADS3.0, Mito-NADS2.0, Mito-NADS3.0) were cloned into the pCDH-CMV-MCS-EF1-Neo vector, and lentivirus was used to Method to prepare HEK293 stably transfected cell line.
  • Probe NADS2.0 is composed of green fluorescent protein mNeoGreen, NAD + binding protein LigA, and bioluminescent protein cpNluc; probe NADS3.0 is composed of red fluorescent protein mScarlet-I, NAD + binding protein LigA, and green fluorescent protein mNeoGreen ( figure 2).
  • NAD + responsive protein does not bind NAD + molecules, the probe structure is in an "open" state, resulting in a longer distance between the resonance energy transfer donor and the acceptor, lower resonance energy transfer efficiency, and the overall probe emits resonance energy transfer Giver of light.
  • NAD + concentration levels can be indicated by measuring the intensity of resonance energy transfer donor and acceptor emission wavelengths within the probe.
  • Example 2 Mito-NADS2.0 and Nuc-NADS2.0 measure NAD + levels in cells treated with Alpha-GPC
  • HEK 293T cells stably expressing Mito-NADS2.0 or Nuc-NADS2.0 were plated in a 96-well white cell culture plate at a cell volume of 10,000 cells/well, DMEM medium (containing a variety of amino acids, vitamins, inorganic salt and high-concentration glucose, without phenol red), the dosage is 100 ⁇ L.
  • the Flex Station3 multifunctional microplate reader was used to detect the emission intensity at wavelengths of 590 nm and 440 nm. According to the detection principle of this probe, the ratio of emission intensity at 590 nm and 440 nm wavelengths indicates the NAD + concentration in living cells.
  • Alpha-GPC at a final concentration of 0.1-10 ⁇ M can significantly reduce the mitochondrial NAD + content; in the presence of 10 nM FK866, adding a final concentration of 10 ⁇ M Alpha-GPC can also significantly reduce the NAD + concentration.
  • Figure 3(B) shows that the final concentration of 5-10 ⁇ M Alpha-GPC can significantly increase the nuclear NAD + level; at the same time, when treated with 10 nM FK866, 1-10 ⁇ M Alpha-GPC can significantly increase the nuclear NAD + concentration.
  • Example 3 Mito-NADS3.0 and Nuc-NADS3.0 measure NAD + levels in cells treated with Alpha-GPC
  • HEK 293T cells stably expressing Mito-NADS3.0 or Nuc-NADS3.0 were plated in a 35 mm diameter cell culture dish at a cell volume of 10 5 cells/mL in DMEM medium (high sugar, no phenol red). ) The dosage is 2 mL.
  • DMEM medium high sugar, no phenol red
  • the dosage is 2 mL.
  • cells were treated with different final concentrations of Alpha-GPC (0 ⁇ M, 5 ⁇ M) in the presence of FK866 (10 nM).
  • the NAD + content of cells was measured using fluorescence microscopy.
  • the fluorescence microscope parameters were set as follows: the excitation wavelength was 488 nm, and the emission wavelengths were 532 nm and 561 nm.

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Abstract

本发明公开了甘油磷酸胆碱在调控细胞NAD +水平和分布中的应用，甘油磷酸胆碱能够特异性调控细胞核和线粒体NAD +水平和分布，其可特异性提高细胞核NAD +水平和分布而降低线粒体NAD +水平和分布，为基于亚细胞NAD +代谢调控的疾病治疗策略提供药物和营养干预方法。

Description

甘油磷酸胆碱在调控细胞NAD+水平和分布中的应用 技术领域
本发明属于生物技术领域，涉及一种化合物的应用，具体而言，是甘油磷酸胆碱在调控细胞烟酰胺腺嘌呤二核苷酸（NAD +）水平和分布中的应用。
背景技术
烟酰胺腺嘌呤二核苷酸（NAD +）对所有生物体均具有重要的功能：作为一种辅酶因子参与数百种氧化还原反应；是数十种调控酶分子如PARPs和SIRTs的底物；参与非典型的RNA-加帽修饰。因而，胞内NAD +对细胞代谢、细胞信号传导和基因表达调控具有重要影响。细胞内NAD +水平在衰老、神经退行、肾损伤、肥胖和糖尿病等众多生理现象中发生动态变化。在真核细胞中，NAD +的合成及消耗具有高度区室化的特点，并广泛分布于细胞质、细胞核、线粒体、内质网和高尔基体。活细胞内区室化的NAD +代谢状态调控了与疾病和代谢紊乱相关的诸多生理过程，是整合能量代谢和信号诱导基因转录的重要机制。研究发现，脂肪生成信号可迅速诱导细胞质NAD +合成酶NMNAT-2的表达，并与细胞核NAD +合成酶NMNAT-1竞争共同底物烟酰胺单核苷酸，导致细胞核NAD +水平急剧下降，使原有NAD +时空分布失衡，并进一步诱导细胞的成脂转录，诱导脂肪细胞分化，而逆转细胞核NAD +下降则可以减少细胞的成脂转录。该细胞器NAD +时空分布的失衡过程在脂质细胞分化和神经母细胞瘤功能调控中有显著作用，重塑细胞器NAD +时空分布有望在调控成脂分化和神经母细胞瘤功能中起积极作用。同时有研究发现，细胞质NAD +通过调节翻译和维持蛋白质稳态在卵巢癌中发挥重要作用。细胞质NMNAT-2的表达在卵巢癌中高度上调，与细胞核NAD +合成产生竞争，导致细胞核NAD +水平下降，NAD +时空分布失衡。同时，NMNAT-2可维持单(ADP-核糖基)转移酶PARP-16的催化活性，而PARP-16可使核糖体蛋白单ADP-核糖基化。NMNAT-2或PARP-16的下调则会导致单ADP-核糖基化的抑制、多核糖体结合的增加、特定mRNA翻译的增强、相关蛋白质产物的聚集以及卵巢癌细胞生长的减少。说明由细胞质NMNAT-2介导的细胞核NAD +水平下降与NAD +时空分布失衡通过调控PARP-16活性在卵巢癌等癌症的蛋白质稳态和代谢异常中起重要作用。综上所述，由于NAD +在线粒体和细胞核的分布状态调控了众多疾病相关过程，开发NAD +细胞器特异性调控分子具有重要意义，是有前景的代谢药物和营养干预策略。
甘油磷酸胆碱（L-α-glycerophosphoryl choline，简称Alpha-GPC）由胆碱、甘油和磷酸盐组成，结构图如图1所示。Alpha-GPC天然存在于人体，是合成神经递质乙酰胆碱的前体，具有重要的营养保健功能和医药用价值。众多学术研究及临床试验表明，Alpha-GPC具有提升认知功能、抗高血脂和保护血管的作用，可用于治疗诸如阿尔兹海默症和精神分裂症等疾病。但目前尚无Alpha-GPC与NAD +的关系的研究报告。
技术问题
为了解决现有技术中的不足，本发明旨在提供甘油磷酸胆碱在调控细胞烟酰胺腺嘌呤二核苷酸水平和分布中的应用。
 技术解决方案
本发明第一方面提供甘油磷酸胆碱在调控细胞烟酰胺腺嘌呤二核苷酸水平和分布比率中的应用。
进一步地，所述甘油磷酸胆碱用于调控活细胞的亚细胞结构烟酰胺腺嘌呤二核苷酸水平和分布。
进一步地，所述亚细胞结构为细胞核或线粒体。
进一步地，所述甘油磷酸胆碱用于提高细胞核烟酰胺腺嘌呤二核苷酸水平和分布，和/或降低线粒体烟酰胺腺嘌呤二核苷酸水平和分布。
本发明第二方面提供甘油磷酸胆碱在制备预防和/或治疗细胞烟酰胺腺嘌呤二核苷酸代谢调控相关疾病的药物组合物中的应用。
进一步地，所述甘油磷酸胆碱用于制备预防和/或治疗亚细胞结构烟酰胺腺嘌呤二核苷酸代谢调控相关疾病的药物组合物。
进一步地，所述甘油磷酸胆碱用于制备预防和/或治疗细胞核烟酰胺腺嘌呤二核苷酸水平和分布降低，和/或线粒体烟酰胺腺嘌呤二核苷酸水平和分布升高相关疾病的药物组合物；
优选地，所述细胞烟酰胺腺嘌呤二核苷酸代谢调控相关疾病包括脂质细胞分化异常、线粒体能量代谢疾病、表现为亚细胞NAD +分布失衡的阿尔兹海默氏病和假性缺氧等。
本发明第三方面提供甘油磷酸胆碱在制备用于调控细胞烟酰胺腺嘌呤二核苷酸水平和分布、抗衰老、延长细胞寿命、辅助降血脂或辅助降血糖的保健品、膳食补充剂、功能性食物、医疗食物或营养补充剂中的应用。
本发明第四方面提供一种调控细胞烟酰胺腺嘌呤二核苷酸水平和分布的药物制剂，所述药物制剂的活性成分包括甘油磷酸胆碱；
优选地，所述药物制剂用于提高细胞核烟酰胺腺嘌呤二核苷酸水平和分布，和/或降低线粒体烟酰胺腺嘌呤二核苷酸水平和分布。
本发明第五方面提供一种预防和/或治疗细胞烟酰胺腺嘌呤二核苷酸代谢调控相关疾病的药物组合物，所述药物组合物的活性成分包括甘油磷酸胆碱；
优选地，所述药物组合物用于预防和/或治疗细胞核烟酰胺腺嘌呤二核苷酸水平和分布降低，和/或线粒体烟酰胺腺嘌呤二核苷酸水平和分布升高相关疾病。
本发明第六方面提供一种保健品、膳食补充剂、功能性食物、医疗食物或营养补充剂，所述保健品、膳食补充剂、功能性食物、医疗食物或营养补充剂的成分包括甘油磷酸胆碱；
优选地，所述保健品、膳食补充剂、功能性食物、医疗食物或营养补充剂用于调控细胞烟酰胺腺嘌呤二核苷酸水平和分布、抗衰老、延长细胞寿命、辅助降血脂或辅助降血糖。
 有益效果
本发明的有益效果为：
本发明首次报道Alpha-GPC能够特异性调控细胞核和线粒体NAD +水平和分布，Alpha-GPC可特异性提高细胞核NAD +水平而降低线粒体NAD +水平，为基于亚细胞NAD +代谢调控的疾病治疗策略提供药物和营养干预方法。
Alpha-GPC不会对人体产生任何急性、亚急性或慢性危害，可用于开发用于调控细胞烟酰胺腺嘌呤二核苷酸水平和分布、抗衰老、延长细胞寿命、辅助降血脂或辅助降血糖的保健品、膳食补充剂、功能性食物、医疗食物或营养补充剂。
 附图说明
图1是化合物Alpha-GPC的结构示意图。
图2（A）是探针NADS2.0和NADS3.0工作示意图。（B）为添加细胞核或线粒体定位肽的探针NADS2.0和NADS3.0在细胞中特异表达示意图。
图3（A）为线粒体特异表达探针NADS2.0的HEK 293T细胞在Alpha-GPC与FK866不同浓度组合处理24 h后，在515 nm和440 nm处发光强度比值图。（B）为细胞核特异表达探针NADS2.0的HEK 293T细胞在Alpha-GPC与FK866不同浓度组合处理24 h后，在515 nm和440 nm处发光强度比值图。所有的P值均是使用非配对双尾Student’s t tests计算得到：*p < 0.05，**p < 0.01；***p < 0.001；****p < 0.0001。
图4（A）是稳转线粒体或细胞核特异表达探针NADS3.0的HEK 293T细胞经过Alpha-GPC处理后的FRET信号显微图。（B）为稳转线粒体或细胞核特异表达探针NADS3.0的HEK 293T细胞经过Alpha-GPC处理后的FRET（RFP/GFP）信号统计直方图。
 本发明的实施方式
为了使本发明的上述目的、特征和优点能够更加明显易懂，下面对本发明的具体实施方式做详细的说明，但不能理解为对本发明的可实施范围的限定。
实施例 1：稳转NAD +分子探针的HEK 293T细胞制备
为了探测细胞中NAD +分子浓度变化，首先需要构建稳定表达NAD +分子探针的HEK 293T细胞。所使用的NAD +分子探针为基于生物发光共振能量转移的探针NADS2.0和基于荧光共振能量转移的探针NADS3.0。分别向这两个NAD +分子探针的氨基酸序列N-端添加细胞核定位肽序列（NTS：DPKKKRKV）和线粒体定位序列（MTS：SVLTPLLLRGLTGSARRLPVPRAKIHSL），获得Nuc-NADS2.0（SEQ ID NO. 1）、Nuc-NADS3.0（SEQ ID NO. 2）和Mito-NADS2.0（SEQ ID NO. 3）、Mito-NADS3.0（SEQ ID NO. 4），实现NAD +分子探针的细胞核及线粒体特异性表达。将这四种探针（Nuc-NADS2.0、Nuc-NADS3.0、Mito-NADS2.0、Mito-NADS3.0）的编码基因克隆至pCDH-CMV-MCS-EF1-Neo载体，利用慢病毒法制备HEK293稳转细胞系。
探针NADS2.0由绿色荧光蛋白mNeoGreen、NAD +结合蛋白LigA、和生物发光蛋白cpNluc组成；探针NADS3.0由红色荧光蛋白mScarlet-I、NAD +结合蛋白LigA、和绿色荧光蛋白mNeoGreen组成(图2)。当NAD +响应蛋白不结合NAD +分子时，探针结构处于“开放”状态，导致共振能量转移给体和受体之间距离较远，共振能量转移效率较低，探针整体发出共振能量转移给体的光。当NAD +响应蛋白结合NAD +分子后，其构象由“开放”转变为“闭合”状态，促使共振能量转移给体和受体靠近，形成较高的共振能量转移效率，探针发出共振能量转移受体的光。因而，可以通过测量探针内共振能量转移给体和受体发射波长强度来指示NAD +浓度水平。
实施例 2：Mito-NADS2.0和Nuc-NADS2.0测量经Alpha-GPC处理后细胞的NAD +水平
分别将稳定表达Mito-NADS2.0或Nuc-NADS2.0的HEK 293T细胞，以10000个/孔的细胞量铺在96孔白色细胞培养板中，DMEM培养基（包含多种氨基酸、维生素、无机盐及高浓度葡萄糖，无酚红）用量为100 μL。在37℃，5 % CO 2条件下培养24 h后，利用不同终浓度的Alpha-GPC（0 μM, 0.1 μM, 0.5 μM, 1 μM, 5 μM, 10 μM）在FK866（NAMPT抑制剂，可有效降低细胞内NAD +水平）不存在或存在（10 nM）的情况下处理细胞。化合物处理细胞24 h后，将DMEM培养基（高糖，无酚红）换成新鲜的含生物发光底物的DMEM培养基（高糖，无酚红，生物发光底物稀释1000-倍）。利用Flex Station3多功能酶标仪检测590 nm和440 nm波长的发射光强。根据该探针的检测原理，590 nm和440 nm波长的发射光强比值指示活细胞中的NAD +浓度。
由图3(A)可知，0.1-10 μM 终浓度Alpha-GPC均能显著降低线粒体NAD +含量；在10 nM FK866存在的情况下，加入终浓度10 μM Alpha-GPC也能显著降低NAD +浓度。图3(B)表明，终浓度5-10 μM Alpha-GPC可显著提升细胞核NAD +水平；同时使用10 nM FK866处理时，1-10 μM Alpha-GPC均能显著增加细胞核NAD +浓度。
实施例 3：Mito-NADS3.0和Nuc-NADS3.0测量经Alpha-GPC处理后细胞的NAD +水平
分别将稳定表达Mito-NADS3.0或Nuc-NADS3.0的HEK 293T细胞，以10 5个/mL的细胞量铺在直径35 mm的细胞培养皿中，DMEM培养基（高糖，无酚红）用量为2 mL。在37℃，5 % CO 2条件下培养24 h后，利用不同终浓度的Alpha-GPC（0 μM, 5 μM）在FK866（10 nM）存在的情况下处理细胞。化合物处理细胞24 h后，利用荧光显微镜测量细胞NAD +含量。荧光显微镜参数设置如下：激发光波长488 nm，发射波长为532 nm和561 nm。用imageJ图像处理软件计算RFP/GFP光强比值。
由图4可知，用5 μM 终浓度Alpha-GPC处理后，细胞核FRET信号显著增高，而线粒体FRET信号显著降低。这表明，细胞核NAD +水平显著增高，而线粒体NAD +浓度则显著降低。

Claims (10)

  1. 甘油磷酸胆碱在调控细胞烟酰胺腺嘌呤二核苷酸水平和分布比率中的应用。
  2. 根据权利要求1所述的应用，其特征在于，所述甘油磷酸胆碱用于调控活细胞的亚细胞结构烟酰胺腺嘌呤二核苷酸水平和分布；
    优选地，所述亚细胞结构为细胞核或线粒体。
  3. 根据权利要求1所述的应用，其特征在于，所述甘油磷酸胆碱用于提高细胞核烟酰胺腺嘌呤二核苷酸水平和分布，和/或降低线粒体烟酰胺腺嘌呤二核苷酸水平和分布。
  4. 甘油磷酸胆碱在制备预防和/或治疗细胞烟酰胺腺嘌呤二核苷酸代谢调控相关疾病的药物组合物中的应用。
  5. 根据权利要求4所述的应用，其特征在于，所述甘油磷酸胆碱用于制备预防和/或治疗亚细胞结构烟酰胺腺嘌呤二核苷酸代谢调控相关疾病的药物组合物。
  6. 根据权利要求5所述的应用，其特征在于，所述甘油磷酸胆碱用于制备预防和/或治疗细胞核烟酰胺腺嘌呤二核苷酸水平和分布降低，和/或线粒体烟酰胺腺嘌呤二核苷酸水平和分布升高相关疾病的药物组合物；
    优选地，所述细胞烟酰胺腺嘌呤二核苷酸代谢调控相关疾病包括脂质细胞分化异常、线粒体能量代谢疾病、表现为亚细胞NAD +分布失衡的阿尔兹海默氏病和假性缺氧。
  7. 甘油磷酸胆碱在制备用于调控细胞烟酰胺腺嘌呤二核苷酸水平和分布、抗衰老、延长细胞寿命、辅助降血脂或辅助降血糖的保健品、膳食补充剂、功能性食物、医疗食物或营养补充剂中的应用。
  8. 一种调控细胞烟酰胺腺嘌呤二核苷酸水平和分布的药物制剂，其特征在于，所述药物制剂的活性成分包括甘油磷酸胆碱；
    优选地，所述药物制剂用于提高细胞核烟酰胺腺嘌呤二核苷酸水平和分布，和/或降低线粒体烟酰胺腺嘌呤二核苷酸水平和分布。
  9. 一种预防和/或治疗细胞烟酰胺腺嘌呤二核苷酸代谢调控相关疾病的药物组合物，其特征在于，所述药物组合物的活性成分包括甘油磷酸胆碱；
    优选地，所述药物组合物用于预防和/或治疗细胞核烟酰胺腺嘌呤二核苷酸水平和分布降低，和/或线粒体烟酰胺腺嘌呤二核苷酸水平和分布升高相关疾病。
  10. 一种保健品、膳食补充剂、功能性食物、医疗食物或营养补充剂，其特征在于，所述保健品、膳食补充剂、功能性食物、医疗食物或营养补充剂的成分包括甘油磷酸胆碱；
    优选地，所述保健品、膳食补充剂、功能性食物、医疗食物或营养补充剂用于调控细胞烟酰胺腺嘌呤二核苷酸水平和分布、抗衰老、延长细胞寿命、辅助降血脂或辅助降血糖。
PCT/CN2022/138175 2022-04-22 2022-12-09 甘油磷酸胆碱在调控细胞nad+水平和分布中的应用 WO2023202099A1 (zh)

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Citations (2)

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CN102525992A (zh) * 2012-03-09 2012-07-04 徐奎 L-α-甘油磷酸胆碱薄膜衣片及其制备方法

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