WO2023236739A1 - 核苷酸混合物在用于防治阿尔茨海默症制剂中的应用 - Google Patents

核苷酸混合物在用于防治阿尔茨海默症制剂中的应用 Download PDF

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WO2023236739A1
WO2023236739A1 PCT/CN2023/094737 CN2023094737W WO2023236739A1 WO 2023236739 A1 WO2023236739 A1 WO 2023236739A1 CN 2023094737 W CN2023094737 W CN 2023094737W WO 2023236739 A1 WO2023236739 A1 WO 2023236739A1
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nts
disease
alzheimer
nucleotide
ump
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李勇
刘睿
徐美虹
陈玉松
曾峥
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陈玉松
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    • 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
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
    • A61K31/7072—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid having two oxo groups directly attached to the pyrimidine ring, e.g. uridine, uridylic acid, thymidine, zidovudine
    • 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
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
    • 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
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
    • A61K31/708—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid having oxo groups directly attached to the purine ring system, e.g. guanosine, guanylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • 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 medicine and health care, and relates to a nucleotide product and its new use. Specifically, it relates to the application of a nucleotide mixture produced by enzymatic degradation using ribonucleic acid as raw material in the preparation of preparations for preventing and treating Alzheimer's disease. .
  • AD Alzheimer's disease
  • MCI mild cognitive impairment
  • EAD early Alzheimer's disease
  • LAD late Alzheimer's disease
  • Amnestic mild cognitive impairment is the first clinical stage of AD, and patients usually experience memory loss and decline; EAD patients gradually develop progressive brain degeneration; LAD patients show severe memory loss, dementia, and behavioral changes. Life and quality of life are seriously affected.
  • AD Alzheimer's disease
  • Nucleotides are the important material basis of heredity, energy metabolism, and signal transduction in the body. Nucleotides can be derived from endogenous synthesis in the human body or exogenous synthesis through biological enzymatic hydrolysis technology. Exogenous nucleotides are indispensable nutrients under specific physiological conditions. In tissues and organs with strong metabolism or when the body is subject to stress, immune challenge, liver damage, starvation, or rapid growth, nucleotides can be absorbed and utilized by the tissue, saving the body's consumption of de novo synthesis or remedial synthesis, thereby optimizing the tissue. Function.
  • enzymatic degradation of nucleic acids into nucleotides in vitro can omit the decomposition process in the body, making it easier to be digested and absorbed by the human body.
  • NTs have become a hot spot in nutrition research due to their extremely high safety and high efficiency, and have been widely used in infant formula milk powder/food, health food/special medical purpose formula food, showing their role in nutritional intervention. important role and broad prospects.
  • exogenous nucleotides obtained by enzymatic hydrolysis technology in preventing and treating Alzheimer's disease.
  • the object of the present invention is to provide a nucleotide mixture with lower molecular weight and fast absorption for use in the preparation of preparations for preventing and treating Alzheimer's disease.
  • the present invention provides the use of a nucleotide mixture in preparing a preparation for preventing and treating Alzheimer's disease, where the nucleotide mixture is a 5'-monophosphate nucleotide mixture.
  • the nucleotide mixture is composed of four or five 5'-mononucleotides or their sodium salt forms, wherein the various nucleotides are converted into the mass of CMP, AMP, UMP, GMP, and IMP acid forms.
  • the ratios are: CMP 23 ⁇ 78%, AMP 6 ⁇ 44%, UMP 7 ⁇ 40%, GMP 7 ⁇ 51%, IMP is 0, or greater than 0 and not higher than 2.5%.
  • the mass ratios of the acid forms of CMP, AMP, UMP, GMP, and IMP from each nucleotide in the nucleotide mixture are respectively: CMP 40-78%, AMP 6-20%, and UMP 7-25%. , GMP 7 ⁇ 20%, IMP 0 ⁇ 1%.
  • the mass ratios of the acid forms of CMP, AMP, UMP, GMP, and IMP from each nucleotide in the nucleotide mixture are respectively: CMP 40-45%, AMP 15-20%, and UMP 20-25% , GMP 15 ⁇ 20%, IMP 0 ⁇ 1%.
  • the preparation is powder, tablet, soft and hard capsule, beverage or oral liquid.
  • the intervention of the 5'-monophosphate nucleotide mixture has a good improvement effect on the learning and memory ability and the decrease in autonomous activity ability related to Alzheimer's disease, and can effectively prevent the learning and memory related to Alzheimer's disease.
  • Memory dysfunction Its mechanism of action is to improve acetylcholine synthesis and utilization disorders, reduce the expression of A ⁇ 1-42 in hippocampal tissue, regulate the expression of CREB pathway proteins in the hippocampus, change the synaptic structure of the hippocampus, and regulate the host's brain function and function through the flora-gut-brain axis. Behavior-related, thereby playing a better role in preventing and treating Alzheimer's disease.
  • the present invention has discovered that the 5'-monophosphate nucleotide mixture can improve the learning and memory impairment related to Alzheimer's disease, effectively prevent the learning and memory dysfunction related to Alzheimer's disease, and has a beneficial effect on the occurrence and development of Alzheimer's disease. It has better preventive and therapeutic effects, and its use in preparing drugs or functional foods for preventing and treating Alzheimer's disease has been discovered.
  • nucleotide mixtures can effectively improve the learning and memory impairment related to Alzheimer's disease, improve the synthesis and utilization of acetylcholine, reduce the expression of A ⁇ 1-42 in hippocampal tissue, and upregulate the CREB pathway and hippocampus in the hippocampus. Synaptic structure-related proteins are expressed and regulate the host's brain function and behavior through the microbiota-gut-brain axis.
  • the results show that the proportioned nucleotide mixture of the present invention produces synergistic effects through multiple ways and ultimately forms a preventive and therapeutic effect on Alzheimer's disease. During the experiment, it was found that the technical effects of different proportions of nucleotide mixtures exist. Significant differences.
  • the nucleotide mixture in this example is composed of four 5'-mononucleotides or their sodium salts in the form of CMP 43wt%, AMP 17wt%, UMP 22wt%, and GMP 18wt%.
  • the nucleotide mixture in this embodiment is composed of five kinds of 5'-mononucleotides or their sodium salts in the form of CMP 23.5wt%, AMP 44wt%, UMP 25wt%, GMP 7wt%, and IMP 0.5wt%.
  • the preparation method is the same as Example 1.
  • the nucleotide mixture in this example is composed of five kinds of 5'-mononucleotides or their sodium salts in the form of CMP 78wt%, AMP 6wt%, UMP 8wt%, GMP 7wt%, and IMP 1wt%.
  • the preparation method is the same as Example 1.
  • the nucleotide mixture in this embodiment is composed of five kinds of 5'-mononucleotides or their sodium salts in the form of CMP 23wt%, AMP 17wt%, UMP 40wt%, GMP 18wt%, and IMP 2wt%.
  • the preparation method is the same as Example 1.
  • the nucleotide mixture in this embodiment is composed of five kinds of 5'-mononucleotides or their sodium salts in the form of CMP 24wt%, AMP 15.5wt%, UMP 7wt%, GMP 51wt%, and IMP 2.5wt%.
  • the preparation method is the same as Example 1.
  • mice The cells used in this example were PC-12 cells derived from rat adrenal pheochromocytoma, purchased from Zhejiang Meisen Cell Technology Co., Ltd.
  • the experimental methods in this example are all conventional methods, and are carried out in accordance with the experimental methods or conditions described in literature in the field or in accordance with the instructions of the kit.
  • the materials, reagents, instruments, etc. used in the following examples can all be obtained from commercial sources.
  • Experimental results are expressed as mean ⁇ standard deviation (x ⁇ SD).
  • Use SPSS software to analyze data for homogeneity of variances.
  • For homogeneous variances use one-way ANOVA; perform appropriate variable transformations for non-normal or uneven variance data to meet normality or homogeneity of variances. Statistics will be performed after the requirements are met; if the variables still do not meet the requirements after conversion, non-parametric tests will be used for statistics.
  • the least significant difference (LSD) method will be used to compare the experimental group and the control group, with P ⁇ 0.05 as the significance standard.
  • the nucleotide mixture used in this example was dissolved in complete cell culture medium at a concentration of 100 ⁇ M.
  • the cells were cultured in a 6-well cell culture plate. After the cells adhered and grew, the cells in different holes were set as normal control group, model control group, and nucleotide mixture group (Examples 1-5 were recorded as NTs mixture 1 and NTs respectively. Mixture 2, NTs mixture 3, NTs mixture 4, NTs mixture 5), single nucleotide control group ( ⁇ -nicotinamide mononucleotide, NMN).
  • NTs mixture 3 NTs mixture 4
  • NTs mixture 5 single nucleotide control group
  • NMN single nucleotide control group
  • cells with good growth status were cultured in normal complete medium
  • the model control group the cells after growth and attachment were cultured in complete medium containing 200 ⁇ mol/L hydrogen peroxide for 4 hours, and the culture containing hydrogen peroxide was discarded.
  • Model animals Three-month-old healthy specific pathogen free (SPF) grade male rapid aging model mouse SAMP8 and model control mouse SAMR1 mice were selected and purchased from the Experimental Animal Center of Peking University Health Science Center (Experimental Animal Production License No. : SCXK (Beijing) 2016-0010; Experimental Animal Use License Number: SYXK (Beijing) 2016-0041). During the experiment, the animals were kept in single cages, the temperature of the animal room was controlled at 22 ⁇ 2°C, the relative humidity was maintained at 50% to 60%, and the day:night light-dark alternation time was 12h:12h.
  • SPPF pathogen free
  • SAMP8 mice are a good animal model for studying the mechanisms of learning and memory dysfunction caused by AD and for evaluating nootropic drugs.
  • the model is relatively stable and has been widely used in research to explore AD-related learning and memory impairment. Therefore, this study selected 3-month-old male SAMP8 mice as an AD mouse model and performed NTs intervention before rapid aging occurred to explore its effect on AD-related memory loss and its possible mechanisms.
  • mice were three months old, each group was given corresponding feed for feeding intervention. During the experiment, the animals drank and ate freely. After the intervention until the mice were 12 months old, behavioral experiments related to learning and memory were conducted; the hippocampus tissue of the mice was collected, rapidly frozen in liquid nitrogen, and then stored in a -80°C refrigerator for later use.
  • mice When the mice were 12 months old (that is, 9 months after NTs intervention), 12 mice from each group were randomly selected for behavioral experiments, including the Morris water maze test. In order to reduce systematic errors and ensure the consistency of observations before and after the experiment, each behavioral experiment was conducted by a fixed person, and the movement of people was reduced and kept quiet throughout the experiment. There should be at least 3 days between each two behavioral experiments to eliminate interference.
  • the platform was randomly placed in the first quadrant and fixed 1 to 1.5cm underwater.
  • the water temperature was maintained at 21°C ⁇ 1°C. It lasted for seven days and was divided into two parts: positioning navigation experiment and space exploration experiment.
  • Orientation navigation test used to measure the learning and memory abilities of mice in the water maze. The experiment lasted 6 days. Before starting the test, place the platform in the center of the first quadrant and fix the platform position unchanged. During daily testing, the mice were gently put into the water from the four quadrants of the east, west, south, and north facing the pool wall, and the time it took for the mice to find the platform from entering the water (escape latency) and swimming distance were recorded. and swimming speed. There is an interval of 5 minutes between each test, and the average of the four results will be entered into the final statistics as the final score of the day. If the animal does not find the platform within 60 s, the experimenter will guide it to the platform and stay on the platform for 10 s, and the escape latency is recorded as 60 s.
  • Spatial exploration experiment used to measure the mouse's memory retention ability of the spatial location of the platform.
  • the platform was removed, and the mice were put into the water from any entry point in the third quadrant.
  • the time from entering the water to when the mice first arrived at the platform was recorded as the escape latency, and the mice within 60 seconds were recorded.
  • the residence time in the target quadrant (the quadrant where the platform is located in the positioning navigation experiment), the swimming distance in the target quadrant, and the number of times the platform is accurately crossed.
  • mice Mouse hippocampal tissue was taken, and the UV colorimetric method was used to determine the hippocampal acetylcholine transferase (ChAT) and acetyl cholinesterase (AChE) activities of mice in each group according to the kit instructions.
  • ChAT hippocampal acetylcholine transferase
  • AChE acetyl cholinesterase
  • Mouse hippocampal tissue was taken, and Western Blot was used to detect amyloid precursor protein (Amyloid Precursor Protein, APP), ⁇ -amyloid protein ( ⁇ -amyloid protein, A ⁇ 1-42), and cyclic adenosine monophosphate response element in the mouse hippocampal tissue.
  • Amyloid Precursor Protein APP
  • ⁇ -amyloid protein ⁇ -amyloid protein, A ⁇ 1-42
  • cyclic adenosine monophosphate response element in the mouse hippocampal tissue.
  • Binding protein cAMP-response element binding protein, CREB
  • protein kinase A C ⁇ subunit p-PKAC ⁇
  • brain-derived neurotrophic factor BDNF
  • postsynaptic density protein 95 Postsynaptic density protein 95, PSD95
  • Ca2+/calmodulin-dependent protein kinase II Calcium/calmodulin-dependent protein kinase II, CaMK II
  • protein kinase C ⁇ subunit Protein kinase C ⁇ subunit, PKC ⁇
  • phosphorylated N-methyl-D- The expression level of aspartate receptor 1 (N-methyl-D-aspartate receptor1, NMDAR1) was measured.
  • the Morris water maze is a relatively objective and accurate method for evaluating animal learning and memory, especially spatial learning and memory functions.
  • animals need to learn repeatedly and recognize the spatial positions of objects around the maze, find the best route in memory to approach the platform in the maze, and find and climb the platform in the shortest time. If the animal's spatial learning and memory ability declines, its escape latency and swimming distance will be prolonged; and the spatial exploration experiment is used to measure the animal's ability to remember the spatial location of the platform after it learns to find the platform.
  • the effect of NTs intervention on the spatial learning and memory of the Morris water maze in SAMP8 mice is shown in Figure 1.
  • the escape latency of mice in the NTs-Free and Normal control groups was significantly greater than that of the NTs-L and NTs-M groups (P ⁇ 0.05), while crossing
  • the number of platforms, target quadrant residence time and target quadrant distance were all significantly smaller than those of the NTs-L and NTs-M groups (P ⁇ 0.05 or P ⁇ 0.01).
  • the results show that the spatial learning and memory abilities of mice in the NTs-Free and Normalcontrol groups decreased significantly at 12 months of age, while NTs intervention can significantly shorten the escape latency of SAMP8 mice, find the platform faster, and increase the number of times they cross the platform.
  • AD Alzheimer's disease
  • ChAT is an important initiating enzyme for cholinergic effects, which can promote the synthesis of ACh from acetyl-CoA and choline in cells.
  • AChE can act on ACh in the synaptic cleft to rapidly hydrolyze it to form acetate and choline, which is the key enzyme that leads to the inactivation of ACh in the body.
  • the normal maintenance of hippocampal cholinergic system function can promote the normal performance of memory-related activities.
  • the training process of various behavioral experiments can cause an increase in hippocampal ACh levels and induce long-term increases in ChAT levels.
  • the effect of NTs intervention on the activity of AChE and ChAT in the hippocampus of SAMP8 mice is shown in Figure 3.
  • the AChE level in the NTs-L group was significantly lower than that in the NTs-Free and Normalcontrol groups (P ⁇ 0.05, P ⁇ 0.01).
  • the H group was significantly lower than the Normal control group (P ⁇ 0.05).
  • the ChAT level in the NTs-L group was significantly higher than the NTs-Free and Normalcontrol groups (P ⁇ 0.05), and the NTs-H group was significantly higher than the NTs-Free group (P ⁇ 0.05).
  • NTs intervention can prevent and treat disorders of acetylcholine synthesis and utilization by reducing AChE activity and increasing ChAT activity in brain tissue, thereby protecting the normal operation of the cholinergic system in normal brain tissue and preventing learning and memory disorders.
  • ⁇ -amyloid protein is recognized as a biomarker of Alzheimer’s disease. ⁇ -amyloid protein is deposited in senile plaques as amyloid fibrils or amorphous aggregates of non-fibrils. This forms the typical brain features of Alzheimer's disease.
  • a ⁇ is produced by the hydrolysis of amyloid precursor protein APP in the body, mainly including A ⁇ 1-42 , A ⁇ 1-40 and A ⁇ 1-43 . Among them, A ⁇ 1-42 is the main type of A ⁇ and the main component of senile plaques in the brains of AD patients.
  • a ⁇ oligomers are neurotoxic and the main cause of the decline in learning and memory in AD patients.
  • large amounts of A ⁇ oligomers induce a series of biochemical changes, leading to a reduction in synaptic plasticity of nerve cells and a decline in learning and memory abilities.
  • soluble A ⁇ oligomers can inhibit the induction and enhancement of synaptic LTP.
  • the concentration of A ⁇ oligomers in the brains of AD patients is 70 times higher than that in the brains of normal controls.
  • SAMP8 mice passed the growth period, excessive soluble A ⁇ was spontaneously produced in the hippocampus along with rapid decline in learning and memory. Similar to AD, it is these soluble A ⁇ that cause the decline of learning and memory in SAMP8 mice.
  • morphological results found that SAMP8 mice had no obvious loss of hippocampal neurons, and hippocampal neuron apoptosis is a typical manifestation of late AD. Therefore, studies believe that SAMP8 mice are a better animal model of early A ⁇ -related learning and memory loss in AD.
  • FIG 3 The effect of NTs intervention on the expression of APP and A ⁇ 1-42 in the hippocampus of SAMP8 mice is shown in Figure 3.
  • the APP protein expression content in the NTs-L group was significantly lower than that in the NTs-Free and Normalcontrol groups (P ⁇ 0.05); A ⁇ 1-
  • the expression content of 42 protein in the NTs-L and NTs-M groups was significantly lower than that in the NTs-Free and Normalcontrol groups, and the difference was significant (P ⁇ 0.05). It shows that NTs can intervene in its potential damaging effects on hippocampal neurons and synaptic plasticity by reducing the deposition of amyloid in brain tissue, and has the potential to slow down the development of learning and memory disorders and reduce the risk of neurodegenerative diseases.
  • a represents the comparison with the NTs-Free group
  • b represents the comparison with the Normal control group
  • * represents the significant difference: P ⁇ 0.05;#:P ⁇ 0.01.
  • AD is accompanied by transcriptional and epigenetic changes in the hippocampus and surrounding cortex, which will eventually lead to abnormal expression of related proteins, thereby inhibiting the long-term potentiation (LTP) process, causing a decrease in synaptic plasticity, and affecting the body's learning and memory.
  • LTP long-term potentiation
  • CREB Cyclic adenosine monophosphate response element binding protein
  • p-CREB Sustained phosphorylation of CREB
  • PKA is one of the major protein kinases upstream of CREB. Activated PKA can directly induce the phosphorylation of CREB, or indirectly induce the activation of CREB by activating ERK.
  • Brain-derived neurotrophic factor (BDNF) is an important downstream target factor regulated by CREB and a very important member of the large family of nerve growth factors (NGFs). It is widely present in the nervous system and is most abundant in the hippocampus.
  • BDNF is released in a dependent manner mediated by NMDAR-CaMKII, acting on the presynaptic and postsynaptic membranes to activate specific receptor tyrosine kinase receptor B (Tyrosine kinase receptor B, TrkB), triggers structural and functional LTP, regulates synaptic plasticity, and affects learning and memory.
  • TrkB specific receptor tyrosine kinase receptor B
  • BDNF can promote the survival, growth and development of various neurons by inducing changes in the structure and function of axonal growth cones, thereby promoting brain development during the growth period and enhancing learning and memory abilities.
  • the effect of NTs intervention on CREB pathway protein expression in the hippocampus of SAMP8 mice is shown in Figure 4.
  • the expression level of p-CREB in the hippocampus of mice in the NTs-L, NTs-M, and NTs-H groups was significantly higher than that in the NTs-Free group, and In NTs-M, it was even higher than that in Normal control and SAMR1 groups, and the difference was significant (P ⁇ 0.05 or P ⁇ 0.01).
  • the expression level of p-PKA ⁇ in the NTs-L and NTs-M groups was significantly higher than that in the NTs-Free, Normal control and SAMR1 groups (P ⁇ 0.05).
  • NTs-L, NTs-M, and NTs-H groups were significantly higher than that in the NTs-Free and Normal control groups (P ⁇ 0.05 or P ⁇ 0.01).
  • the results show that NTs can prevent the reduction of phosphorylated PKA, CREB and BDNF levels in the hippocampus of SAMP8 mice.
  • the possible mechanism is that NTs increase the activity of protein kinases such as PKA and increase the phosphorylation level of the Ser133 site of the transcription factor CREB; in addition, it may also directly regulate the CREB signaling pathway in hippocampal nerve cells to exert its neuroprotective effect.
  • NTs increase the activity of CREB in the hippocampus of AD mice by directly regulating the intracellular protein kinase pathway, and increase the expression of neuroprotective target genes such as BDNF.
  • these target genes can promote the activation of CREB.
  • the long-term existence of a virtuous cycle may cause changes in the hippocampal synaptic structure of mice with long-term NTs intervention, increase synaptic transmission efficiency, and thereby promote learning and memory.
  • a represents the comparison with the NTs-Free group
  • b represents the comparison with the Normal control group
  • c and d represent the comparison with the NTs-L and NTs-M groups respectively
  • * represents the significant difference: P ⁇ 0.05 ;#: P ⁇ 0.01.
  • Ca 2+ /calmodulin-dependent protein kinase II (CaMK II) is considered to be a necessary condition for inducing LTP formation and related dendritic spine structural plasticity, and its role in signal transmission between neurons, development of neural circuits and Plays a key role in cognitive function.
  • Inhibition of CaMKII activity occurs during AD, leading to a decrease in dependent NMDAR receptor function, damaging synaptic plasticity and causing cognitive impairment related to neurodegenerative diseases.
  • NMDAR is a glutamate ligand and voltage dual-gated ion channel that plays a very important role in the production of synaptic LTP.
  • the NMDAR1 subunit is an essential component of the NMDAR ion channel and is its functional subunit. After some researchers knocked out the NMDAR1 subunit in the CA1 area of rats, the LTP mediated by NMDAR in this area was significantly weakened, and the accuracy of spatial learning and memory in rats was impaired. In recent years, studies have proven that PKC is closely related to synaptic plasticity and the formation of LTP, and the activation of PKC is an important condition for the production of LTP.
  • Postsynaptic density protein is a specialized area composed of a variety of proteins located under the postsynaptic membrane of the central nervous system. It shows an increased density under an electron microscope and plays a role in mediating and integrating synaptic signal transmission. and play an important role in learning and memory.
  • PSD95 is a special cytoplasmic protein in PSD and is also the main framework component in the PSD structure.
  • the effect of NTs intervention on the expression of synaptic structural proteins in the hippocampus of SAMP8 mice is shown in Figure 5.
  • the expression level of p-CAMKII in the mouse hippocampus in the NTs-L group was significantly higher than that in the NTs-Free, Normalcontrol and SAMR1 groups (P ⁇ 0.05orP ⁇ 0.01).
  • the expression level of PKC ⁇ in the NTs-L and NTs-H groups was significantly higher than that in the NTs-Free and Normalcontrol groups (P ⁇ 0.05).
  • the expression level of p-NMDAR1 in the NTs-L and NTs-M groups was significantly higher than that in the NTs-Free, Normalcontrol and SAMR1 groups, and in the NTs-H group it was significantly higher than that in the NTs-Free group (P ⁇ 0.05 or P ⁇ 0.01).
  • the expression level of PSD95 in the NTs-Free and Normalcontrol groups was lower than that in the other groups, but there was no significant statistical difference (P>0.05).
  • the above research results show that NTs intervention can improve the expression of Ca 2+ regulation-related enzymes in the brain tissue of SAMP8 mice, and the overall improvement effect of the NTs-L group is better than that of the other groups, thereby alleviating calcium regulation abnormalities and regulating synaptic plasticity.
  • a represents the comparison with the NTs-Free group
  • b represents the comparison with the Normal control group
  • c and d represent the comparison with the NTs-L and NTs-M groups respectively
  • * represents the difference is significant: P ⁇ 0.05 ;#: P ⁇ 0.01.
  • Gut microbiota metabolites and their effects on host neurochemistry as well as pathogenic infections may increase the risk of Alzheimer's disease.
  • the researchers compared the differences in intestinal flora, brain metabolism, cerebrovascular function, and cognitive behavior between AD and young mice, and found that the composition of the intestinal flora in AD mice changed: alpha diversity increased, Firmicutes / Bacteroidetes ratio increased; the content of various amino acids and fatty acids related to AD in brain metabolites increased, the blood-brain barrier function was damaged, learning and memory ability decreased, and anxiety increased.
  • Gut flora may regulate the host's brain function and behavior through the flora-gut-brain axis, including changes in cognitive behavior. Nutritional intervention based on the flora-gut-brain axis will become AD An important way of prevention and treatment.
  • the effect of NTs on the intestinal flora of mice was studied. As shown in Figure 7, at the phylum level, the abundance of Proteobacteria, Patescibacteria and Verrucomicrobia was high in the NTs-Free group. In other groups, the abundance of Bacteroidetes was less than that of other groups.
  • the NTs-L group and NTs-M group had the highest Firmicutes content and lower Bacteroidetes content; the NMN group had Firmicutes, Proteobacteria, The content of Patescibacteria is the lowest and the content of Bacteroidetes is the highest. It can be seen that compared with the NTs-Free group, NTs intervention can moderately reduce the contents of Proteobacteria, Patesciba and Verrucomicrobia. Compared with the NTs group, the NMN group intervention It can reduce the content of Firmicutes and increase the content of Bacteroidetes.
  • ANOVA analysis of variance analysis found that at the genus level, the bacterial genera that have a negative impact on the body, Such as uncultured_bacterium_o_Clostridiales, uncultured_bacterium_f_Desulfovibrionaceae, Tyzzerella, Ralstonia, Rumiclostridium_5, Lachnospiraceae_UCG-006, Lachnospiraceae Lachnospiraceae_FCS020_group, Anaerovorax and Bacteroides all have the highest relative abundance in NTs-Free, indicating that NTs can significantly affect the intestinal flora of mice, especially increasing the abundance level of pathogenic bacteria.
  • NTs can significantly affect Amino acid metabolism (amino acid metabolism), Carbohydrate metabolism (carbohydrate metabolism), Enery metabolism (energy metabolism), Membrane transport (membrane transport), Metabolism of cofactors and vitamins (cofactor and vitamin metabolism), Nucleotide metabolism (nucleotide metabolism), Replication and repair (replication and repair), Signal transduction (signal transduction), Translation (translation) and other metabolic pathways.
  • NTs may affect the relative abundance of bacterial flora closely related to the above metabolism-related pathways, thereby regulating the intestinal flora of mice.
  • NTs intervention can increase the decrease in bacterial abundance caused by AD, improve the bacterial structure (beneficial/harmful bacteria ratio), and reduce the relative abundance of bacterial groups that have a negative impact on the body.
  • the mechanism may be It regulates the intestinal flora of mice by affecting the relative abundance of bacterial flora closely related to metabolism-related pathways, and then may regulate the host's brain function and behavior through the flora-gut-brain axis, including recognition. Behavioral changes.
  • the present invention uses the rapidly aging SAMP8 mouse model to study the effect of NTs on preventing Alzheimer's disease-related memory loss and its possible mechanism.
  • NTs intervention has a certain protective effect on the learning and memory abilities of mice, and can effectively prevent learning and memory dysfunction related to Alzheimer's disease.
  • the mechanism by which NTs works may be related to improving acetylcholine synthesis and utilization disorders, reducing the expression of A ⁇ 1-42 in hippocampal tissue, regulating the CREB pathway and hippocampal synaptic structure-related protein expression in the hippocampus, and regulating through the flora-gut-brain axis. It is related to the host's brain function and behavior, thereby achieving early prevention and treatment of Alzheimer's disease.

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Abstract

本发明公开了核苷酸混合物在制备用于防治阿尔茨海默症制剂中的应用，属于医药保健领域。所述核苷酸混合物由四种或五种5'-单核苷酸或者其钠盐形式组成，各种核苷酸折合成CMP、AMP、UMP、GMP、IMP酸型的质量比分别为：CMP 23～78％、AMP 6～44％、UMP 7～40％、GMP 7～51％、IMP为0、或大于0且不高于2.5％。本发明发现摄入核苷酸能够有效改善阿尔茨海默症相关的学习记忆能力减退，改善乙酰胆碱合成及利用障碍，降低海马组织Aβ1-42表达，调节海马CREB通路和海马突触结构相关蛋白表达，并通过菌群-肠-脑轴来调节宿主的脑功能及行为，从而对阿尔茨海默症起到较好的防治作用。

Description

核苷酸混合物在用于防治阿尔茨海默症制剂中的应用 技术领域
本发明属于医药保健领域，涉及一种核苷酸产品及其新用途，具体涉及以核糖核酸为原料经酶法降解生产的核苷酸混合物在制备用于防治阿尔茨海默症制剂中的应用。
背景技术
阿尔茨海默症(Alzheimer's disease，AD)，又称老年痴呆症，是以认知和学习记忆障碍为典型特征的老年常见病。据世界卫生组织统计至2050年，全球受老年痴呆症影响的人数将从5000万人上升至1.5亿。我国AD的患病率(60岁以上)约为4.9％，患病人数占全世界病例的1/4。AD的进展可以分为三个阶段：轻度认知障碍(mild cognitive impairment，MCI)、早期阿尔兹海默症(EAD)及晚期阿尔兹海默症(LAD)。遗忘性的轻度认知障碍是AD的第一临床阶段，患者通常出现记忆缺失与减退；EAD患者逐渐出现进行性脑退化；LAD患者则表现为严重的记忆力减退、痴呆、行为改变，其日常生活及生命质量严重受到侵害。AD发病率及死亡率仅次于肿瘤、心脏病及脑卒中，位居第四位，已成为了降低老年患者生命质量最常见的病症之一。现阶段尚缺乏针对AD的有效防治手段，现有治疗药物并不能阻止疾病的发展或者逆转神经功能的衰退，且多数存在副作用。鉴于营养活性成分具有安全、多效、可预防性干预的优势特点，积极寻找筛选安全、有效能够早期防治AD的候选物，开发辅助改善老年记忆的活性物质，对提高老年人生活质量，减轻社会负担，具有重大的科学意义、经济和社会效益。
核苷酸(Nucleotide，NTs)是机体重要的遗传、能量代谢、信号转导的物质基础。核苷酸可以来源于人体内源性合成以及通过生物酶解技术等外源性合成。外源性核苷酸在特定的生理条件下是不可缺少的营养成分。在代谢旺盛的组织器官或者当机体受到应激、免疫挑战、肝损伤、饥饿以及快速生长的情况下，核苷酸能被组织吸收利用，节省机体从头合成或者补救合成的消耗，从而可以优化组织功能。此外，在体外通过酶解方式将核酸降解成为核苷酸后可以省略体内的分解过程，更加容易被人体消化吸收。目前，NTs以其极高的安全性和高效性特点成为营养研究的热点，并已被广泛用于婴儿配方乳粉/食品，保健食品/特殊医学用途配方食品中，显示出其在营养干预中的重要作用和广阔前景。但目前对于酶解技术获得的外源性核苷酸在防治阿尔茨海默症方面的作用研究尚未见报道。
发明内容
本发明的目的在于提供一种分子量较低、吸收快的核苷酸混合物在制备用于防治阿尔茨海默症制剂中的应用。
为实现上述发明目的，本发明采用以下技术方案：
本发明提供了核苷酸混合物在制备用于防治阿尔茨海默症制剂中的应用，所述核苷酸混合物为5’-单磷酸核苷酸混合物。
进一步地，所述核苷酸混合物由四种或五种5’-单核苷酸或者其钠盐形式组成，其中各种核苷酸折合成CMP、AMP、UMP、GMP、IMP酸型的质量比分别为：CMP 23～78％、AMP 6～44％、UMP 7～40％、GMP 7～51％、IMP为0、或大于0且不高于2.5％。
进一步地，所述核苷酸混合物中各核苷酸折合成CMP、AMP、UMP、GMP、IMP酸型的质量比分别为：CMP 40～78％、AMP 6～20％、UMP 7～25％、GMP 7～20％、IMP为0～1％。
进一步地，所述核苷酸混合物中各核苷酸折合成CMP、AMP、UMP、GMP、IMP酸型的质量比分别为：CMP 40～45％、AMP 15～20％、UMP 20～25％、GMP 15～20％、IMP为0～1％。
进一步地，所述制剂为粉剂、片剂、软硬胶囊、饮料或口服液。
进一步地，5’-单磷酸核苷酸混合物干预对阿尔茨海默症相关的学习记忆能力及自主活动能力减退等均具有较好的改善作用，可以有效预防与阿尔茨海默病相关的学习记忆功能障碍。其作用机制与改善乙酰胆碱合成及利用障碍，降低海马组织中Aβ1-42的表达、调节海马内CREB通路蛋白表达，改变海马突触结构及通过菌群-肠-脑轴来调节宿主的脑功能及行为有关，从而对阿尔茨海默症起到较好的防治作用。
本发明相对于现有技术具有的有益效果如下：
本发明发现了5’-单磷酸核苷酸混合物可以改善阿尔茨海默症相关的学习记忆能力减退、有效预防阿尔茨海默病相关的学习记忆功能障碍，对阿尔茨海默症发生发展具有较好的防治作用，并发现了其在制备防治阿尔茨海默症的药物或功能性食品中的用途。
动物实验证实，摄入核苷酸混合物可有效改善阿尔茨海默症相关的学习记忆能力减退，改善乙酰胆碱合成及利用障碍，降低海马组织中Aβ1-42的表达，上调海马内CREB通路和海马突触结构相关蛋白表达，并通过菌群-肠-脑轴来调节宿主的脑功能及行为。结果表明本发明所述配比的核苷酸混合物是通过多种途径产生协同效果最终形成对阿尔茨海默症的防治作用，并且实验过程中发现不同配比的核苷酸混合物技术效果均存在明显差异。
附图说明
图1为NTs干预对SAMP8小鼠Morris水迷宫空间学习记忆的影响(n＝12)；
图2为NTs干预对SAMP8小鼠海马AChE、ChAT活力的影响(n＝10)；
图3为NTs干预对SAMP8小鼠海马APP和Aβ1-42表达的影响(n＝3)；
图4为NTs干预对SAMP8小鼠海马CREB通路蛋白表达的影响(n＝3)；
图5为NTs干预对SAMP8小鼠海马突触结构蛋白表达的影响(n＝3)；
图6为NTs干预对SAMP8小鼠肠道菌群分布的影响(n＝8)。
具体实施方式
下面结合具体实施方案对本发明作进一步的说明，这些实例应被理解为仅是举例说明，而非以任何方式限制本发明的范围。
实施例1
1.本实施例核苷酸混合物是由四种5’-单核苷酸或者其钠盐的形式按照CMP 43wt％、AMP 17wt％、UMP 22wt％、GMP 18wt％比例混合。
2.具体制备方式如下：
(1)将四种5’-单核苷酸或者其钠盐分别进行检测，合格后备用。
(2)将质检合格的四种5’-单核苷酸或者其钠盐过60目筛备用。
(3)按比例计算称取所需的各单核苷酸样品量，全部加入后进行总混，混合时间不低于40分钟。所得样品常温保存。
实施例2
本实施例核苷酸混合物是由五种5’-单核苷酸或者其钠盐的形式按照CMP 23.5wt％、AMP 44wt％、UMP 25wt％、GMP 7wt％、IMP 0.5wt％比例混合。制备方法同实施例1。
实施例3
本实施例核苷酸混合物是由五种5’-单核苷酸或者其钠盐的形式按照CMP 78wt％、AMP 6wt％、UMP 8wt％、GMP 7wt％、IMP 1wt％比例混合。制备方法同实施例1。
实施例4
本实施例核苷酸混合物是由五种5’-单核苷酸或者其钠盐的形式按照CMP 23wt％、AMP 17wt％、UMP 40wt％、GMP 18wt％、IMP 2wt％比例混合。制备方法同实施例1。
实施例5
本实施例核苷酸混合物是由五种5’-单核苷酸或者其钠盐的形式按照CMP 24wt％、AMP 15.5wt％、UMP 7wt％、GMP 51wt％、IMP 2.5wt％比例混合。制备方法同实施例1。
实施例6
一、材料与方法
1.样品：上述实施例1-5中所得的核苷酸混合物样品。
2.实验细胞：本实施例中所使用的细胞均为来源于大鼠肾上腺嗜铬细胞瘤的PC-12细胞，购自浙江美森细胞科技有限公司。
3.神经退行性细胞模型的建立：细胞培养于含1％青霉素/链霉素10％胎牛血清的DMEM高糖培养基中，在5％CO2培养箱37℃饱和湿度条件下培养。神经退行性细胞模型的建立如下：细胞生长贴壁后，不同浓度的过氧化氢进行干预，浓度分别为50μmol/L、100μmol/L、200μmol/L、400μmol/L、800μmol/L，孵育4h后弃掉含有过氧化氢的培养基，加入正常完全培养基(含1％青霉素/链霉素10％胎牛血清的DMEM高糖培养基)孵育24h，使用CCK-8细胞活性试剂盒检测细胞活性。选择细胞活力约为正常细胞活力的50％的过氧化氢浓度应用于后续实验，即选择了200μmol/L的过氧化氢浓度进行后续实验。
4.实验方法：
本实施例中的实验方法，如无特殊说明，均为常规方法，按照本领域内的文献所描述的实验方法或条件或者按照试剂盒说明书进行。下述实施例中所用的材料、试剂、仪器等均可从商业途径得到。实验结果用均数±标准差(x±SD)表示。运用SPSS软件对数据进行行方差齐性分析，方差齐采用单因素方差分析(one-way ANOVA)；对非正态或方差不齐的数据进行适当的变量转换，满足正态性或者方差齐性要求后进行统计；若变量转换后仍未达到要求，采用非参数检验进行统计，实验组与对照组两两组间比较采用最小显著差异法(LSD)，以P<0.05为差异显著性标准。
本实施例中所使用的核苷酸混合物以100μM的浓度溶于细胞完全培养基中。
将细胞培养于6孔细胞培养板，待细胞贴壁生长后将不同孔细胞设置为正常对照组、模型对照组、核苷酸混合物组(实施例1-5分别依次记作NTs混合物1、NTs混合物2、NTs混合物3、NTs混合物4、NTs混合物5)、单一核苷酸对照组(β-烟酰胺单核苷酸，NMN)。正常对照组将生长状态良好的细胞培养于正常完全培养基中；模型对照组将生长贴壁后的细胞用含有200μmol/L过氧化氢的完全培养基培养4h，弃掉含有过氧化氢的培养基后，加入正常完全培养基孵育24h；5个核苷酸混合组将生长贴壁后的细胞用含有200μmol/L过氧化氢的完全培养基培养4h，弃掉含有过氧化氢的培养基后，分别加入含有100μmol/L核苷酸混合物的完全培养基培养24h；单一核苷酸对照组将生长贴壁后的细胞用含有200μmol/L过氧化氢的完全培养基培养4h，弃掉含有过氧化氢的培养基后，分别加入含有100μmol/L单一核苷酸的完全培养基培养24h。使用CCK-8细胞活性试剂盒检测细胞活力。
二、实验结果
NTs对神经退行性变PC-12细胞活力的影响
与正常对照组比较，模型对照组细胞内细胞活力显著降低(P<0.05)。与模型对照组比较，NMN细胞活力有所升高，但明显低于外源NTs混合物，且外源NTs混合物中NTs混合物1效果最佳(P<0.05)(表1)。
表1外源NTs对NTs对神经退行性变PC-12细胞活力的影响

注：#与正常对照组比较差异有统计学意义，*与模型对照组比较差异有统计学意义，P<0.05；#：P<0.01。
实施例7
一、材料与方法
1.样品：上述实施例1中所得的核苷酸混合物样品。
2.模型动物：选用三月龄健康无特定病原体(Specific pathogen free，SPF)级雄性快速老化模型鼠SAMP8和模型对照鼠SAMR1小鼠，购自北京大学医学部实验动物中心(实验动物生产许可证号：SCXK(京)2016-0010；实验动物使用许可证号：SYXK(京)2016-0041)。实验期间动物行单笼饲养，动物室温度控制在22±2℃，相对湿度保持为50％～60％，昼：夜明暗交替时间为12h：12h。
SAMP8小鼠是研究AD引起的学习记忆功能障碍发生机制以及评价益智药物的良好动物模型，模型较为稳定，被广泛应用到探索AD相关的学习记忆减退的研究中。因此，本研究选取了3月龄的雄性SAMP8小鼠作为AD小鼠模型，在其快速老化发生之前进行NTs干预，探索其对AD相关记忆减退的作用及其可能机制。
3.实验分组与剂量：SAMP8小鼠适应性喂养一周后，按体重随机分为5组(n＝20)：无核苷酸组(纯化饲料，NTs-Free)、普通对照组(基础饲料，Normal control)、NTs低剂量干预组(0.3g/kg+基础饲料，NTs-L)、NTs中剂量干预组(0.6g/kg+基础饲料，NTs-M)、NTs高剂量干预组(1.2g/kg+基础饲料，NTs-H)，并设立SAMR1模型对照组(基础饲料，n＝20)。核苷酸混合物按照上述不同剂量掺入基础饲料中进行干预。自小鼠三月龄起，各组给予对应饲料进行喂养干预，实验期间动物自由饮水与进食。干预至小鼠12月龄时，进行学习记忆相关行为学实验；并留取小鼠海马组织，于液氮中急速冷冻后储存在-80℃冰箱内待用。
4.实验方法
4.1行为学实验
在小鼠12月龄时(即NTs干预9个月后)，每组随机选12只进行行为学实验，包括Morris水迷宫实验。为减小系统误差、保证实验前后观察的一致性，各行为学实验均由固定人员负责、实验全程减少人员走动、保持安静。每两个行为学实验之间至少间隔3天，以消除干扰。
Morris水迷宫实验(Morris water maze)
实验时将平台随机置于第一象限并固定于水下1～1.5cm，水温保持在21℃±1℃。共历时7天，分为定位航行实验和空间探索实验两部分。
定位航行实验：用于测量小鼠在水迷宫中的学习和记忆能力。实验历时6天。测试开始前，将平台放置于第一象限中央，并固定平台位置不变。每日测试时，小鼠分别从东、西、南、北4个象限面向池壁轻轻放入水中，记录小鼠从入水到找到平台所需的时间(逃避潜伏期，escape latency)、游泳路程和游泳速度。每次测试间隔5min，4次成绩的平均值作为当日最终成绩进入最后统计。如果动物在60s内未找到平台，则将由实验者将其引导至平台并在平台上停留10s，逃避潜伏期记为60s。
空间探索实验：用于测量小鼠对平台空间位置的记忆保持能力。实验第7天，撤去平台，将小鼠从第三象限的任选一个入水点放入水中，记录小鼠从入水到第一次到达平台所在位置的时间记为逃避潜伏期，记录60s内小鼠在目标象限(定位航行实验中平台所在象限)的停留时间、目标象限游泳路程及准确穿越平台所在位置的次数。
4.2脑胆碱能系统相关酶活力的检测
取小鼠海马组织，采用紫外比色法，根据试剂盒说明书测定各组小鼠海马乙酰胆碱转移酶(Choline acetyltransferase，ChAT)及乙酰胆碱酯酶(Acetyl cholinesterase，AChE)活力。
4.3 Western Blot检测小鼠海马相关蛋白表达水平
取小鼠海马组织，采用Western Blot对小鼠海马组织中：淀粉样前体蛋白(Amyloid Precursor Protein，APP)、β淀粉样蛋白(β-amyloid protein,Aβ1-42)、环磷腺苷效应元件结合蛋白(cAMP－response element binding protein，CREB)、蛋白激酶A Cβ亚基(p-PKACβ)、脑源性神经营养因子(Brain-derived neurotrophic factor，BDNF)、突触后致密蛋白95(Postsynaptic density protein 95,PSD95)、Ca2+/钙调蛋白依赖性蛋白激酶Ⅱ(Calcium/calmodulin-dependent protein kinaseⅡ，CaMKⅡ)、蛋白激酶Cγ亚单位(Protein kinase Cγsubunit，PKCγ)、磷酸化N-甲基-D-天冬氨酸受体1(N-methyl-D-aspartate receptor1，NMDAR1)的表达水平进行测定。
4.4肠道菌群结构及主要功能菌属鉴定
收集小鼠盲肠内容物，对菌群16S rRNA基因V3区进行扩增和纯化，使用引物对为27F(5’-ID-AGAGTTTGATCCTGGCTCAG-3’)和533R(5’-ID-TTACCGCGGCTGCTGGCAC-3’)。之后进行454焦磷酸测序，测序在Roche 454GS FLX平台上进行。对预处理合格的全部序列使用在线工具进行对比后，导入ARB工具，根据序列相似性水平(取97％)划分操作分类单位(Operational taxonomic unit，OTU)，选取丰度最高的序列作为该OTU的代表序列，评估测序量和各个文库的物种多样性、物种丰富度。采用UniFrac分析方法对菌群结构差异进行分析，多样品间进行相似度比对及组间差异显著性分析，筛选出有显著差异的一组变量操作分类单位，即造成各组动物菌群结构差异的关键影响因子。
5统计学分析
各组数据分别录入Excel数据库，使用SPSS 24.0软件(SPSS，Inc.，Chicago，IL，USA)对数据进行分析。连续变量数据用均数±标准差表示，采用单因素方差分析(One-way analysis of variance，ANOVA)对结果进行分析，采用最小显著性差异法(Least-significant difference，LSD)进行组间的统计检验；变量转换后仍未达到正态或方差齐的数据，采用Kruskal-Wallis检验。对于记数资料，采用卡方检验进行不同组之间差异的统计学分析。确定P＜0.05具有统计学意义。
二、实验结果
1.NTs对SAMP8小鼠空间学习记忆能力的影响
Morris水迷宫是一种评价动物学习记忆，尤其是空间学习记忆功能较为客观而准确的方法。在Morris水迷宫定位航行实验中，动物需要通过反复学习、认识迷宫周围物体的空间位置，寻找记忆里接近迷宫中平台的最佳路线，用最短时间找到并爬上平台。若动物的空间学习记忆能力下降，其逃避潜伏期和游泳距离将延长；而空间探索实验用于测量动物学会寻找平台后，对平台空间位置记忆的能力。
NTs干预对SAMP8小鼠Morris水迷宫空间学习记忆的影响结果如图1所示，NTs-Free、Normal control组小鼠逃避潜伏期显著大于NTs-L、NTs-M组(P＜0.05)，同时穿越平台次数、目标象限停留时间及目标象限路程均明显小于NTs-L、NTs-M组(P＜0.05or P＜0.01)。结果说明NTs-Free、Normalcontrol组小鼠在12月龄时空间学习记忆能力显著下降，而NTs干预可明显缩短SAMP8小鼠的逃避潜伏期，寻找到平台的速度更快，穿越平台所在位置的次数增加，目标象限停留的时间延长。表明NTs能够有效预防SAMP8小鼠空间学习记忆能力的减退。图1中，a代表与NTs-Free组相比；b代表与Normal control组相比；*代表差异具有显著性：P<0.05；#：P<0.01。
2.NTs对SAMP8小鼠胆碱能系统酶活力的影响
AD患者认知功能衰退程度与海马组织及皮质中胆碱能系统异常有关。AD患者脑内多表现出ChAT及AChE水平的异常，其诱导了胆碱能神经元缺失及胆碱能神经纤维的退行性改变的发生发展。乙酰胆碱(acetylcholine，ACh)是大脑学习记忆与认知能力的重要神经传导介质，胆碱能神经递质的减少可直接导致学习记忆障碍，甚至直接增加罹患老年痴呆症的风险。ChAT是胆碱能发挥作用的重要启动酶，其能促进细胞乙酰辅酶A与胆碱合成ACh。AChE可作用于突触间隙中的ACh使其迅速水解形成乙酸盐及胆碱，是导致机体内ACh失活的关键酶。海马胆碱能系统功能的正常维持可促进记忆相关活动的正常进行，各种行为学实验的训练过程可引起海马ACh水平的升高，并诱发ChAT的长期水平提升。
NTs干预对SAMP8小鼠海马AChE、ChAT活力的影响结果如图3所示，AChE水平在NTs-L组要明显低于NTs-Free和Normalcontrol组(P＜0.05，P＜0.01)，在NTs-H组明显低于Normal control组(P＜0.05)。ChAT水平在NTs-L组要明显高于NTs-Free和Normalcontrol组(P＜0.05)，在NTs-H组明显高于NTs-Free组(P＜0.05)。表明NTs干预可以通过降低脑组织内AChE活性及增加ChAT的活性，对乙酰胆碱合成及利用障碍具有防治作用，从而起到保护正常脑组织胆碱能系统正常运作，预防学习记忆障碍的作用。
3.NTs对SAMP8小鼠海马Aβ生成的影响
β淀粉样蛋白(β-amyloid protein,Aβ)被公认为是阿尔兹海默症的生物标志物，老年斑中β淀粉样蛋白沉积为淀粉样蛋白原纤维或非原纤维的无定形聚集体，由此形成阿尔茨海默病的典型脑特征。Aβ在体内由淀粉样前体蛋白APP经水解产生，主要有Aβ1-42、Aβ1-40和Aβ1-43。其中Aβ1-42是Aβ的主要类型，也是构成AD病人脑内老年斑的主要成分。
Aβ的神经毒性是多种因素导致AD发病的共同通路，能引起一系列的细胞内生理和生化改变。近年来，对Aβ研究的重心从不溶性的淀粉样斑块逐渐转移到可溶性的寡聚体上。Aβ寡聚体是发挥神经毒性，造成AD病人学习记忆下降的主要原因。在AD的早期阶段，大量的Aβ寡聚体诱导一系列的生化改变，导致神经细胞突触可塑性的降低，学习记忆能力的下降。在海马切片的体外培养和一些体内实验均发现可溶性Aβ寡聚体能够抑制突触LTP的诱导和强化。AD病人脑部Aβ寡聚体的浓度是正常对照大脑的70倍。SAMP8小鼠在度过生长期后，伴随着学习记忆的快速减退海马内自发生成过量的可溶性Aβ。和AD类似，正是这些可溶性Aβ引起SAMP8小鼠的学习记忆的减退。同时形态学结果发现SAMP8小鼠没有明显的海马神经元的丢失，而海马神经元的凋亡是AD晚期的典型表现。因此研究认为SAMP8小鼠是比较好的AD早期Aβ相关学习记忆减退的动物模型。
NTs干预对SAMP8小鼠海马APP和Aβ1-42表达的影响结果如图3所示，APP蛋白表达含量在NTs-L组要明显低于NTs-Free及Normalcontrol组(P＜0.05)；Aβ1-42蛋白表达含量在NTs-L及NTs-M组要明显低于NTs-Free及Normalcontrol组，差异具有显著性(P＜0.05)。表明NTs可通过减少脑组织内淀粉样蛋白的沉积，来干预其潜在的对海马神经元及突触可塑性的侵害作用，具有减缓学习记忆障碍发展，降低神经退行性疾病发生风险的潜力。图3中，a代表与NTs-Free组相比；b代表与Normal control组相比；*代表差异具有显著性：P<0.05；#：P<0.01。
4.NTs对SAMP8小鼠海马CREB通路的影响
AD伴随着海马及周围皮质中转录和表观遗传学的改变，最终会导致相关蛋白的表达异常，进而抑制长时程增强(LTP)进程，造成突触可塑性的下降，对机体的学习及记忆能力造成影响。环磷腺苷效应元件结合蛋白(CREB)是长期突触可塑性维持的重要转录因子，其在磷酸化后可被各种酶激活。CREB的持续磷酸化(p-CREB)被认为是增强LTP所必需的，在控制神经元功能中起到重要作用，如参与调控神经元分化及学习与记忆有关基因的转录，辅助细胞增殖与生存等。PKA是CREB上游主要的蛋白激酶之一。活化的PKA能够直接诱导CREB的磷酸化，或者通过激活ERK从而间接的诱导CREB的活化。脑源性神经营养因子(BDNF)是CREB调控的重要下游靶因子，是神经生长因子(Nerve growthfactors，NGFs)大家族中非常重要的一员。广泛存在于神经系统，其中以海马中含量最为丰富。BDNF对学习记忆的影响可能通过以下方式：(1)BDNF在NMDAR-CaMKII介导下依赖性释放，作用于突触前膜和突触后膜，激活特异性受体酪氨酸激酶受体B(Tyrosine kinase receptor B，TrkB)，引发结构和功能LTP，调节突触可塑性，影响学习与记忆。(2)BDNF能够通过诱导轴突生长锥结构和功能的变化，促进多种神经元的存活和生长发育，从而促进生长期的脑部发育，增强学习记忆能力。
NTs干预对SAMP8小鼠海马CREB通路蛋白表达的影响如图4所示，小鼠海马组织p-CREB表达水平在NTs-L、NTs-M、NTs-H组明显高于NTs-Free组，且在NTs-M还要高于Normal control及SAMR1组，差异具有显著性(P＜0.05or P＜0.01)。p-PKAβ表达水平在NTs-L、NTs-M组明显高于NTs-Free、Normal control及SAMR1组(P＜0.05)。BDNF表达水平在NTs-L、NTs-M、NTs-H组要显著高于NTs-Free及Normal control组(P＜0.05or P＜0.01)。结果表明NTs能够预防SAMP8小鼠海马内磷酸化的PKA、CREB及BDNF水平的降低。其机制可能为NTs使PKA等蛋白激酶活性增高，转录因子CREB的Ser133位点磷酸化水平增高；此外，还可能直接调节海马神经细胞内CREB信号途径，发挥其神经保护作用。可见，NTs通过直接调节细胞内蛋白激酶通路，增加AD小鼠海马内CREB的活性，增加BDNF等具有神经保护作用的靶基因的表达，反过来这些靶基因又能够促进CREB的活化，当这种良性循环长期存在，可能会使NTs长期干预小鼠的海马神经突触结构发生变化，突触传递效能增加，进而促进学习记忆。图4中，a代表与NTs-Free组相比；b代表与Normal control组相比；c、d分别代表与NTs-L、NTs-M组相比；*代表差异具有显著性：P<0.05；#：P<0.01。
5.NTs对SAMP8小鼠海马突触结构蛋白表达的影响
为进一步探索AD和NTs对突触形态结构的影响，我们对一些突触相关结构蛋白表达进行测定。采用Western blot方法检测了突触结构相关蛋白PSD95、CAMKII、NMDAR1和PKCγ的表达情况。
在学习与记忆过程中，神经元细胞处于兴奋状态，其中NMDAR的激活会导致细胞内Ca2+浓度的增加，进而触发一系列依赖钙的信号级联反应激活。其中，Ca2+/钙调蛋白依赖性蛋白激酶Ⅱ(CaMKⅡ)被认为是诱导LTP形成及相关神经树突棘结构可塑性的必要条件，其在神经元之间的信号传递、神经电路的发育及认知功能当中起到了关键作用。AD过程中会发生CaMKⅡ活性的抑制，进而导致依赖性NMDAR受体功能的下降，破坏突触可塑性并造成神经退行性疾病相关的认知障碍的出现。NMDAR是一种谷氨酸的配基和电压双门控离子通道，在突触LTP的产生过程中，起着非常重要的作用。NMDAR1亚单位是组成NMDAR离子通道的必需配件，是其功能性亚基。有研究者将大鼠CA1区NMDAR1亚基敲除后，该区由NMDAR介导的LTP明显消弱，大鼠空间学习记忆的精确性受到损害。近年来研究证明，PKC与突触可塑性及LTP的形成密切相关，PKC的激活是LTP产生的重要条件。突触后致密物(postsynaptic density protein,PSD)是位于中枢神经系统突触后膜下由多种蛋白构成的特化区，在电子显微镜下呈密度增高影，在介导和整合突触信号传递以及学习记忆中发挥重要的作用。PSD95是PSD中的一种特殊的胞质内蛋白质，也是PSD结构中的主要框架成分。
NTs干预对SAMP8小鼠海马突触结构蛋白表达的影响结果如图5所示，小鼠海马组织p-CAMKII表达水平在NTs-L组明显要高于NTs-Free、Normalcontrol及SAMR1组(P＜0.05orP＜0.01)。PKCγ表达水平在NTs-L、NTs-H组要明显高于NTs-Free、Normalcontrol组(P＜0.05)。p-NMDAR1表达水平在NTs-L、NTs-M组要明显高于NTs-Free、Normalcontrol及SAMR1组，在NTs-H组要明显高于NTs-Free组(P＜0.05or P＜0.01)。PSD95表达水平在NTs-Free、Normalcontrol组要低于其它各组，但无明显统计学差异(P>0.05)。以上研究结果表明，NTs干预可以改善SAMP8小鼠的脑组织Ca2+调节相关酶的表达，且NTs-L组整体改善效果要优于其它各组，从而缓解钙调节异常，调控突触可塑性相关蛋白表达，进而改善AD相关学习记忆功能障碍。图5中：a代表与NTs-Free组相比；b代表与Normal control组相比；c、d分别代表与NTs-L、NTs-M组相比；*代表差异具有显著性：P<0.05；#：P<0.01。
5.NTs对SAMP8小鼠菌群-肠-脑轴的影响
肠道菌群代谢产物及其对宿主神经化学的影响以及病原体感染可能增加阿尔兹海默症的发病风险。研究人员对比了AD和年轻小鼠在肠道菌群、脑代谢、脑血管功能、认知行为方面的差异，发现AD小鼠肠道菌群构成发生改变：α多样性增加、厚壁菌门/拟杆菌门的比例上升；脑代谢产物中与AD相关的多种氨基酸和脂肪酸含量增加，血脑屏障功能受损，学习记忆能力下降，焦虑现象增加。在健康人中，肠道革兰氏阴性杆菌产生的脂多糖(的免疫组化信号在脑实质中聚集成块，而在AD患者中，75％的LPS信号聚集在DAPI染色的细胞核的周围；提示在AD患者的脑中大量存在着来源于胃肠道菌群的促炎症信号，随着退行性疾病，这些毒素分子可能从胃肠道中“漏”进全身循环，最终进入脑。对阿尔兹海默症患者、轻度认知障碍患者及健康人的粪便菌群多样性及组成进行比较发现，在轻度认知障碍阶段(阿尔兹海默症的前期)，肠道菌群已发生与疾病阶段相似的变化。肠道菌群有可能通过菌群-肠-脑轴来调节宿主的脑功能及行为，包括认知行为的改变，基于菌群-肠-脑轴进行营养干预将成为AD防治的重要途径。针对NTs对小鼠肠道菌群的影响进行了研究，如图7所示，在门水平上，NTs-Free组变形菌门、Patescibacteria菌门和疣微菌门丰度高于其它各组，拟杆菌门丰度少于其它各组。NTs-L组和NTs-M组厚壁菌门含量最高、拟杆菌门含量较少；NMN组厚壁菌门、变形菌门、Patescibacteria菌门含量最少，拟杆菌门含量最高。可见，与NTs-Free组相比，NTs干预可适度降低变形菌门、Patesciba菌门和疣微菌门含量。与NTs组相比，NMN组干预可降低厚壁菌门含量，增加拟杆菌门含量。为了更准确的了解各组间肠道菌群结构的差异，我们在属水平上进行分析，并结合Alpha多样性分析，结果发现NTs-Free组Ace值和Chao1值均最小，表明NTs-Free组小鼠粪便的细菌丰度较小。而Simpson指数与Shannon指数均为NTs最大，NTs-Free最小。因此，NTs-Free组的小鼠粪便物种多样性最高，而NTs-Free则最低，由此表明NTs可以增加小鼠肠道菌群的多样性。ANOVA(方差分析)分析发现，在属水平上，对机体有负面影响的菌属，如梭菌目未培养菌(uncultured_bacterium_o_Clostridiales)、脱硫弧菌科未培养菌(uncultured_bacterium_f_Desulfovibrionaceae)、Tyzzerella、劳尔氏菌属(Ralstonia)、Ruminiclostridium_5菌属、毛螺菌科(Lachnospiraceae_UCG-006)、毛螺菌科(Lachnospiraceae_FCS020_group)、Anaerovorax以及拟杆菌属(Bacteroides)均在NTs-Free中相对丰度最大，表明NTs能显著影响小鼠的肠道菌群，尤其增加了致病菌的丰度水平。以上结果表明，在无核苷酸组中，对机体有负面影响的菌群相对丰度显著性增加，因此有目的地补充外源核苷酸对调节肠道菌群，维护机体健康至关重要。通过KEGG代谢途径的组成及差异分析发现，NTs的添加能显著影响Amino acid metabolism(氨基酸代谢)、Carbohydrate metabolism(碳水化合物代谢)、Enery metabolism(能量代谢)、Membrane transport(膜转运)、Metabolism of cofactors and vitamins(辅助因子和维生素代谢)、Nucleotide metabolism(核苷酸代谢)、Replication and repair(复制和修复)、Signal transduction(信号转导)、Translation(翻译)等代谢通路。NTs可能是影响了与以上代谢相关通路关系密切的菌群的相关丰度，从而达到调节小鼠肠道菌群。综上，我们可知NTs干预则能够增加AD引起的菌群丰度水平的下降，改善菌群结构(有益菌/有害菌比)，减少对机体有负面影响的菌群相对丰度，其机制可能是通过影响与代谢相关通路关系密切的菌群的相关丰度，从而达到调节小鼠肠道菌群的作用，进而可能通过菌群-肠-脑轴来调节宿主的脑功能及行为，包括认知行为的改变。图6中，A：NTs-Free组；B：Normal control组；C：NTs-L组；D：NTs-M组；E：NTs-H组；F：SAMR1组。
三、实验结论
本发明采用快速老化SAMP8小鼠模型，研究了NTs预防阿尔茨海默病相关的记忆减退的作用及其可能机制。研究发现NTs干预对小鼠的学习记忆能力等均具有一定的保护作用，可以有效预防阿尔茨海默病相关的学习记忆功能障碍。NTs发挥作用的机制可能与改善乙酰胆碱合成及利用障碍，降低海马组织中Aβ1-42的表达，调节海马内CREB通路和海马突触结构相关蛋白表达，及通过菌群-肠-脑轴来调节宿主的脑功能及行为有关，从而实现对阿尔茨海默症起到早期防治的作用。
对于任何熟悉本领域的技术人员而言，在不脱离本发明技术方案范围情况下，都可利用上述揭示的技术内容对本发明技术方案作出许多可能的变动和修饰，或修改为等同变化的等效实施例。因此，凡是未脱离本发明技术方案的内容，依据本发明的技术实质对以上实施例所做的任何简单修改、等同变化及修饰，均应仍属于本发明技术方案保护的范围内。

Claims (5)

  1. 核苷酸混合物在用于制备防治阿尔茨海默症制剂中的应用。
  2. 根据权利要求1所述的应用，其特征在于，所述核苷酸混合物由四种或五种5’-单核苷酸或者其钠盐形式组成，各种核苷酸折合成CMP、AMP、UMP、GMP、IMP酸型的质量比分别为：CMP 23～78％、AMP 6～44％、UMP 7～40％、GMP 7～51％、IMP为0、或大于0且不高于2.5％。
  3. 根据权利要求2所述的应用，其特征在于，所述核苷酸混合物中各核苷酸折合成CMP、AMP、UMP、GMP、IMP酸型的质量比分别为：CMP40～78％、AMP 6～20％、UMP 7～25％、GMP 7～20％、IMP为0～1％。
  4. 根据权利要求1所述的应用，其特征在于，所述制剂为粉剂、片剂、软硬胶囊、饮料或口服液。
  5. 根据权利要求1～4中任意一项所述的应用，其特征在于，5’-单磷酸核苷酸混合物能够有效改善阿尔茨海默症的学习记忆能力减退，调节乙酰胆碱合成及利用障碍，降低海马组织中Aβ1-42的表达，上调海马内CREB通路和海马突触结构相关蛋白表达，并通过菌群-肠-脑轴来调节宿主的脑功能及行为，从而对阿尔茨海默症起到防治作用。
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