WO2024020843A1 - 瓜氨酸或其药学上可接受的盐在制备提高免疫力或预防或治疗焦虑症药物中的应用 - Google Patents

瓜氨酸或其药学上可接受的盐在制备提高免疫力或预防或治疗焦虑症药物中的应用 Download PDF

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WO2024020843A1
WO2024020843A1 PCT/CN2022/108199 CN2022108199W WO2024020843A1 WO 2024020843 A1 WO2024020843 A1 WO 2024020843A1 CN 2022108199 W CN2022108199 W CN 2022108199W WO 2024020843 A1 WO2024020843 A1 WO 2024020843A1
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citrulline
pharmaceutically acceptable
matrix metalloproteinase
acceptable salt
present
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PCT/CN2022/108199
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English (en)
French (fr)
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刘兴国
杨亮
阮子峰
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中国科学院广州生物医药与健康研究院
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Priority to PCT/CN2022/108199 priority Critical patent/WO2024020843A1/zh
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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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • 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
    • A61P25/22—Anxiolytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants

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  • the present invention relates to the field of medicine. Specifically, the present invention relates to the use of citrulline or its pharmaceutically acceptable salts in the preparation of drugs for improving immunity or preventing or treating anxiety disorders.
  • Anxiety disorders and anxiety symptoms associated with mental illness are a type of neuropsychiatric disorder characterized by episodic or persistent anxiety and tension. In recent years, the incidence of anxiety disorders has increased year by year, seriously affecting the quality of life of patients. People who suffer from anxiety for a long time will develop many somatic symptoms, such as sleep disorders, upset, emotional problems, gastrointestinal dysfunction, back pain, unexplained body pain, eating problems, and even excretion problems. These appear Abnormalities will lead to low immune function, resulting in decreased resistance.
  • the present invention aims to solve at least one of the technical problems existing in the prior art to at least a certain extent.
  • the present invention proposes the use of citrulline or its pharmaceutically acceptable salts in the preparation of medicines, methods for improving the activity of matrix metalloproteinase 7, methods for up-regulating the expression of genes encoding matrix metalloproteinase 7, and methods for improving the activity of matrix metalloproteinase 7.
  • Immunity or a pharmaceutical composition for preventing or treating anxiety disorders, a method for preventing or treating anxiety disorders or improving immunity, citrulline or a pharmaceutically acceptable salt thereof is important for the prevention or treatment of anxiety disorders and improving immunity Research value and clinical application value.
  • the invention provides the use of citrulline or a pharmaceutically acceptable salt thereof in the preparation of a medicament.
  • the medicine is used to prevent or treat anxiety disorders or improve immunity.
  • the invention proposes a method for improving matrix metalloproteinase 7 activity.
  • the method includes contacting the matrix metalloprotease with citrulline or a pharmaceutically acceptable salt thereof.
  • the present invention proposes a method for up-regulating the expression of a gene encoding matrix metalloproteinase 7.
  • the method includes: co-culturing citrulline or a pharmaceutically acceptable salt thereof with cells or tissues containing a gene encoding matrix metalloproteinase 7.
  • the present invention provides a pharmaceutical composition for improving immunity or preventing or treating anxiety disorders.
  • the pharmaceutical composition comprises citrulline or a pharmaceutically acceptable salt thereof.
  • the present invention provides a method for preventing or treating anxiety disorders or improving immunity.
  • the method includes: administering citrulline or a pharmaceutically acceptable salt thereof to the subject.
  • Figure 1 shows the immunohistochemistry of MMP7 in the small intestine of wild-type and mtDNA mutant mice according to Example 1 of the present invention
  • Figure 2 shows the open field experiment of wild-type and mtDNA mutant mice according to Embodiment 1 of the present invention, and statistics of the average speed and frequency of mice entering the central area (n ⁇ 3, *p ⁇ 0.05, independent t-test );
  • Figure 3 shows the small intestinal metabolite detection diagram of wild-type and mtDNA mutant mice according to Example 1 of the present invention (n ⁇ 4, *p ⁇ 0.05, independent t-test);
  • Figure 4 shows the immunohistochemistry pattern of MMP7 in the small intestine of mtDNA mutant mice after administration of citrulline according to Example 2 of the present invention
  • the present invention proposes the use of citrulline or its pharmaceutically acceptable salts in the preparation of medicines, methods for improving the activity of matrix metalloproteinase 7, methods for up-regulating the expression of genes encoding matrix metalloproteinase 7, and methods for improving immunity or Pharmaceutical compositions for preventing or treating anxiety disorders, methods for preventing or treating anxiety disorders, or improving immunity will be described in detail below respectively.
  • citrulline or its pharmaceutically acceptable salts in the preparation of medicines
  • the invention provides the use of citrulline or a pharmaceutically acceptable salt thereof in the preparation of a medicament.
  • the medicine is used to prevent or treat anxiety disorders or improve immunity.
  • POLG mutant mice have a D257A mutation at the position of POLG exonuclease, which causes the exonuclease activity to be lost, resulting in proofreading defects during mtDNA replication and causing mtDNA mutations. It is a recognized mouse model for studying mtDNA mutations. , the mtDNA mutant mouse model will suffer from low immunity and anxiety symptoms, and the low immunity is reflected in the reduced expression of matrix metalloproteinase 7 (MMP7). MMP7 mainly cleaves defensin precursors to produce functional defensins, which regulate intestinal mucosal reactions through antibacterial responses and maintain intestinal-intestinal microbial balance. Reduced MMP7 expression will lead to intestinal flora imbalance in mice.
  • MMP7 matrix metalloproteinase 7
  • the inventors conducted a metabolomics study on the small intestine of an mtDNA mutant mouse model and found that citrulline specifically decreased in the mouse model. Furthermore, by administering citrulline to the mtDNA mutant mouse model, the expression of MMP7 was significantly increased. This shows that citrulline can increase the expression of MMP7 and help improve the body's immunity. At the same time, anxiety symptoms in mtDNA mutant mice were alleviated.
  • citrulline and its pharmaceutically acceptable salts can further help prevent or Treatment of Alzheimer's disease, Parkinson's disease, cancers such as breast cancer, lung cancer, nasopharyngeal cancer, liver cancer, stomach cancer, esophageal cancer, colorectal cancer, pancreatic cancer, melanoma, skin cancer, prostate cancer, cervical cancer, leukemia, Thyroid cancer, lymphoma, bladder cancer, kidney cancer, uterine cancer, ovarian cancer, gallbladder cancer, oral cancer, laryngeal cancer, bone cancer, testicular cancer or brain cancer, etc.
  • cancers such as breast cancer, lung cancer, nasopharyngeal cancer, liver cancer, stomach cancer, esophageal cancer, colorectal cancer, pancreatic cancer, melanoma, skin cancer, prostate cancer, cervical cancer, leukemia, Thyroid cancer, lymphoma, bladder cancer, kidney cancer, uterine cancer, ovarian cancer, gallbladder cancer, oral cancer, laryngeal cancer,
  • pharmaceutically acceptable refers to molecular entities and compositions that are physiologically tolerable when administered to humans and generally do not produce allergic or similar inappropriate reactions, such as gastrointestinal upset, dizziness, and the like.
  • pharmaceutically acceptable as used herein means approved by a federal regulatory agency or a state government or listed in the United States Pharmacopeia or other generally recognized pharmacopeia for use in animals, more particularly in humans.
  • the medicament is used to prevent or treat intestinal flora imbalance.
  • citrulline or its pharmaceutically acceptable salts can increase the expression of MMP7, thereby helping to prevent or treat intestinal flora imbalance.
  • the drug is used to increase the activity of matrix metalloproteinase 7 or to up-regulate the expression of genes encoding matrix metalloproteinase 7.
  • the drug targets the brain-gut axis.
  • the brain-gut axis refers to the two-way information exchange network between the brain and the intestines, which broadly includes neural interaction pathways, neuroendocrine and neuroimmune pathways, intestinal microbiota, etc.
  • the inventor detected and analyzed the metabolites of mice with low immunity and found that citrulline decreased in the small intestine, and the small intestine is directly related to the brain-gut axis. After administration of citrulline to mice, the mice's low immunity was alleviated, which indicates that the target of citrulline is the brain-gut axis.
  • the drug is administered orally or via enema.
  • citrulline can improve immunity by targeting the brain-gut axis. Therefore, oral or enema administration can act on the brain-gut axis and better exert its efficacy.
  • the frequency and dosage of administration of the drug of the present invention can be determined by a number of factors including the type of disease to be treated, the route of administration, the patient's age, sex, weight and severity of the disease, and the type of drug as the active ingredient. .
  • the daily dose may be divided into 1 dose, 2 doses, or multiple doses in a suitable form to be administered once, twice, or multiple times throughout the time period, as long as a therapeutically effective amount is achieved .
  • the invention proposes a method for improving matrix metalloproteinase 7 activity.
  • the method includes contacting matrix metalloproteinase 7 with citrulline or a pharmaceutically acceptable salt thereof.
  • citrulline or a pharmaceutically acceptable salt thereof By contacting citrulline or a pharmaceutically acceptable salt thereof with matrix metalloproteinase 7, the activity of matrix metalloproteinase 7 can be improved, thereby facilitating the scientific research or clinical application of matrix metalloproteinase 7.
  • the present invention proposes a method for up-regulating the expression of a gene encoding matrix metalloproteinase 7.
  • the method includes: co-culturing citrulline or a pharmaceutically acceptable salt thereof with cells or tissues containing a gene encoding matrix metalloproteinase 7. Under conditions suitable for cell or tissue culture, citrulline can effectively increase the expression of genes encoding matrix metalloproteinase 7, thus contributing to the scientific research and clinical application of matrix metalloproteinase 7 and its encoding genes.
  • compositions for improving immunity or preventing or treating anxiety disorders comprising:
  • the present invention provides a pharmaceutical composition for improving immunity or preventing or treating anxiety disorders.
  • the pharmaceutical composition comprises citrulline or a pharmaceutically acceptable salt thereof.
  • the pharmaceutical composition further includes: pharmaceutically acceptable excipients.
  • pharmaceutically acceptable carriers may include buffers, preservatives, analgesics, solubilizers, isotonic agents, and stabilizers.
  • pharmaceutically acceptable carriers may include bases, excipients, lubricants, and preservatives.
  • the pharmaceutical composition of the present invention can be prepared into various dosage forms in combination with the above-mentioned pharmaceutically acceptable carriers.
  • the pharmaceutical compositions may be prepared in unit dosage form, such as in single-dose ampoules, such as in multi-dose containers. Pharmaceutical compositions may also be prepared as solutions, suspensions, tablets, pills, capsules and depots.
  • excipients and diluents suitable for pharmaceutical formulations may include: lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol , starch, arabic rubber, alginate, gel, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl paraben, Talc, magnesium stearate and mineral oil.
  • the excipients of the present invention may also include fillers, anticoagulants, lubricants, moisturizers, fragrances and preservatives.
  • the present invention provides a method for preventing or treating anxiety disorders or improving immunity.
  • the method includes: administering citrulline or a pharmaceutically acceptable salt thereof to the subject.
  • the subject of the present invention is not strictly limited and can be a human or other animal, such as a cow, a horse, a rat, a rabbit, a pig, etc.
  • the administration method is oral administration or enema.
  • Antigen microwave repair Use PBS to dilute the EDTA antigen retrieval solution (50 ⁇ , pH9) to 1 ⁇ . Soak the slices in the 1 ⁇ EDTA antigen retrieval solution. Use the microwave oven to heat it at 100 high heat for 10 minutes and 50 low heat for 10 minutes. Take out the glass. slice and let cool naturally. After cooling, the sections were soaked three times in PBS solution. 5 minutes each time. Use a tissue pen to draw a hydrophobic circle around the tissue block. The next step will be carried out using the two-step immunohistochemistry kit (Cat. No.: E-IR-R217-18mL) from Ilarite.
  • E-IR-R217C 3% H2O2
  • E-IR-R217C normal goat serum
  • MMP7 antibody Cat. No.: 3801S
  • E-IR-R217A normal goat serum
  • E-IR-R217B Polyperoxidase-anti-Mouse/Rabbit IgG
  • DAB substrate solution For every 1mL of E-IR-R217E (DAB substrate solution), add 50 ⁇ L of E-IR-R217D (DAB concentrate), mix evenly to form a DAB working solution, add 50 ⁇ L of DAB working solution dropwise into the hydrophobic ring, and keep away from light at room temperature. Incubate for 5 minutes. Wash with tap water for 5 minutes to stop color development.
  • mice were subjected to the open field experiment. First, place the mouse in the center of a 50 ⁇ 50 ⁇ 50cm (length ⁇ width ⁇ height) chamber for 10 minutes, record with a camera located above the square box, and operate with Noulds EthoVision 9.0 software. Between tests, clean the arena with 75% alcohol. During the open field experiment, all behaviors including the average speed and frequency of entering the central area were recorded and analyzed to evaluate the anxiety levels of 8-month-old wild-type and mtDNA mutant mice.
  • mice Weigh 200mg L-citrulline and add it to 250mL SPF grade animal drinking water, mix thoroughly and dissolve.
  • the weight of 8-month-old mtDNA mutant mice was weighed, and the mice were evenly divided into two groups according to their weight.
  • One group was given 250 mL SPF grade animal drinking water (control group), and the other group was given 200 mg L-citrulline.
  • 250mL SPF grade animal drinking water (experimental group). 14 days of uninterrupted water supply. After 14 days of uninterrupted water supply, the MMP levels in the small intestine were detected through immunohistochemical experiments, and the anxiety levels of the mice were detected through open field experiments. .
  • references to the terms “one embodiment,” “some embodiments,” “an example,” “specific examples,” or “some examples” or the like means that specific features are described in connection with the embodiment or example. , structures, materials or features are included in at least one embodiment or example of the invention. In this specification, the schematic expressions of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the specific features, structures, materials or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, those skilled in the art may combine and combine different embodiments or examples and features of different embodiments or examples described in this specification unless they are inconsistent with each other.

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Abstract

本发明提出了瓜氨酸或其药学上可接受的盐在制备药物中的用途、提高基质金属蛋白酶7活性的方法、上调编码基质金属蛋白酶7的基因表达量的方法、用于提高免疫力或者预防或治疗焦虑症的药物组合物、预防或治疗焦虑症和提高免疫力的方法。

Description

瓜氨酸或其药学上可接受的盐在制备提高免疫力或预防或治疗焦虑症药物中的应用 技术领域
本发明涉及医药领域。具体地，本发明涉及瓜氨酸或其药学上可接受的盐在制备提高免疫力或预防或治疗焦虑症药物中的应用。
背景技术
动物或人类在幼龄、体弱、疾病、应激、病原感染等情况下，容易导致免疫力降低，进而发生继发感染(细菌或病毒混合感染)，引起炎性反应(红、热、肿、痛等)和肠道功能紊乱等。目前，对于感染治疗，传统又普遍使用的防治策略是使用抗生素和激素类抗炎药(如氢化可的松、地塞米松等)，虽能有效控制感染性炎症和非感染性炎症，但持续使用会引起多种副作用：如水盐代谢和糖、脂肪、蛋白质代谢的严重紊乱，并引起肾上腺皮质功能衰退、消化系统并发症或加重感染等。
焦虑症以及精神疾病伴发的焦虑症状是一类发作性或持续性焦虑和紧张情绪为主要特征的神经精神疾病。近年来焦虑症的发病率逐年上升，严重影响患者的生存质量。长期处在焦虑情绪之中的人，会产生很多躯体化症状，比如睡眠障碍、心烦、情绪问题、胃肠功能失调、后背疼痛、身上不明原因串痛、饮食问题，甚至排泄问题，这些出现异常之后，都会导致免疫功能低下，从而导致抵抗力下降。
目前，提高免疫力和预防或治疗焦虑症的药物仍有待研究。
发明内容
本发明旨在至少在一定程度上解决现有技术中存在的技术问题至少之一。为此，本发明提出了瓜氨酸或其药学上可接受的盐在制备药物中的用途、提高基质金属蛋白酶7活性的方法、上调编码基质金属蛋白酶7的基因表达量的方法、用于提高免疫力或者预防或治疗焦虑症的药物组合物、预防或治疗焦虑症或提高免疫力的方法，瓜氨酸或其药学上可接受的盐对于焦虑症的预防或治疗以及提高免疫力具有重要的研究价值和临床应用价值。
在本发明的一个方面，本发明提出了瓜氨酸或其药学上可接受的盐在制备药物中的用途。根据本发明的一个实施例，所述药物用于预防或治疗焦虑症或提高免疫力。
在本发明的另一方面，本发明提出了一种提高基质金属蛋白酶7活性的方法。根据本发明的实施例，所述方法包括：将基质金属蛋白酶与瓜氨酸或其药学上可接受的盐接触。
在本发明的又一方面，本发明提出了一种上调编码基质金属蛋白酶7的基因表达量的 方法。根据本发明的实施例，所述方法包括：将瓜氨酸或其药学上可接受的盐与含有编码基质金属蛋白酶7的基因的细胞或者组织共培养。
在本发明的又一方面，本发明提出了一种用于提高免疫力或者预防或治疗焦虑症的药物组合物。根据本发明的实施例，所述药物组合物包含瓜氨酸或其药学上可接受的盐。
在本发明的又一方面，本发明提出了一种预防或治疗焦虑症或提高免疫力的方法。根据本发明的实施例，所述方法包括：向受试者施用瓜氨酸或其药学上可接受的盐。
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
图1显示了根据本发明实施例1的野生型和mtDNA突变小鼠的小肠MMP7免疫组化图；
图2显示了根据本发明实施例1的野生型和mtDNA突变小鼠的旷场实验，并统计小鼠进入中央区域的平均速度和频率检测(n≥3，*p＜0.05，independent t-test)；
图3显示了根据本发明实施例1的野生型和mtDNA突变小鼠的小肠代谢物检测图(n≥4，*p＜0.05，independent t-test)；
图4显示了根据本发明实施例2的mtDNA突变小鼠给药瓜氨酸后小肠MMP7免疫组化图；
图5显示了根据本发明实施例2的mtDNA突变小鼠给药瓜氨酸后的旷场实验，并统计mtDNA突变小鼠进入中央区域的平均速度和频率检测(n＝2，*p＜0.05，independent t-test)。
具体实施方式
下面详细描述本发明的实施例。下面描述的实施例是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
本发明提出了瓜氨酸或其药学上可接受的盐在制备药物中的用途、提高基质金属蛋白酶7活性的方法、上调编码基质金属蛋白酶7的基因表达量的方法、用于提高免疫力或者预防或治疗焦虑症的药物组合物、预防或治疗焦虑症或提高免疫力的方法，下面将分别对 其进行详细描述。
瓜氨酸或其药学上可接受的盐在制备药物中的用途
在本发明的一个方面，本发明提出了瓜氨酸或其药学上可接受的盐在制备药物中的用途。根据本发明的实施例，所述药物用于预防或治疗焦虑症或提高免疫力。
POLG突变的小鼠在POLG核酸外切酶的位置有一个D257A突变使其核酸外切酶活性丧失，导致mtDNA复制过程中的校正缺陷，引起mtDNA突变，是一个公认的研究mtDNA突变的小鼠模型，mtDNA突变小鼠模型会出现免疫力低下和焦虑症状，免疫力低下体现在基质金属蛋白酶7(MMP7)表达量降低。MMP7主要是剪切防御素前体，从而产生有功能的防御素，通过抗菌反应来调控肠道粘膜反应，维持肠道-肠道微生物的平衡。MMP7表达量降低，将导致小鼠出现肠道菌群失调。
进一步地，发明人对mtDNA突变小鼠模型的小肠进行代谢物组学研究，发现小鼠模型体内瓜氨酸特异性下降。进一步地，通过给予mtDNA突变小鼠模型瓜氨酸，MMP7表达量显著升高。由此表明瓜氨酸可以提高MMP7表达量，有助于提高机体免疫力。同时，mtDNA突变小鼠的焦虑症状有所缓解。
人体免疫功能的稳定是维持机体内环境稳定的关键，很多疾病的发生均与免疫力低下相关，瓜氨酸及其药学上可接受的盐在提高免疫力的基础上，进一步有助于预防或者治疗阿尔兹海默病、帕金森病、癌症，例如乳腺癌、肺癌、鼻咽癌、肝癌、胃癌、食道癌、大肠癌、胰腺癌、黑色素瘤、皮肤癌、前列腺癌、宫颈癌、白血病、甲状腺癌、淋巴瘤、膀胱癌、肾癌、子宫体癌、卵巢癌、胆囊癌、口腔癌、喉癌、骨癌、睾丸癌或脑癌等。
术语“药学上可接受的”是指当给人施用时生理上可耐受的并且一般不产生过敏或相似不适当的反应，例如肠胃不适、眩晕等的分子实体和组合物。优选地，本文所用的术语“药学上可接受的”是指联邦监管机构或国家政府批准的或美国药典或其他一般认可的药典上列举的在动物中、更特别在人体中使用的。
根据本发明的实施例，所述药物用于预防或治疗肠道菌群失调。如前所述，瓜氨酸或其药学上可接受的盐可以提高MMP7表达量，进而有助于预防或治疗肠道菌群失调。
根据本发明的实施例，所述药物用于提高基质金属蛋白酶7活性或者上调编码基质金属蛋白酶7的基因表达量。
根据本发明的实施例，所述药物靶向脑-肠轴。脑-肠轴(brain-gut axis)指的是大脑与肠道间的双向信息交流网络，广义上包括神经交互通路、神经内分泌和神经免疫途径、肠道微生物群等。发明人对免疫力低下的小鼠代谢物进行检测分析，发现小肠瓜氨酸下降，而小肠与脑肠轴直接相关。通过给予小鼠瓜氨酸后，小鼠免疫力低下得以缓解，由此，表 明瓜氨酸的作用靶点为脑-肠轴。
根据本发明的实施例，所述药物的给药方式为口服或者灌肠。如前所述，瓜氨酸可以通过靶向脑-肠轴以提高免疫力，因此，采用口服或者灌肠的给药方式，可以作用于脑-肠轴，更好地发挥药效。
本发明药物的给药频率和剂量可以通过多个相关因素被确定，该因素包括要被治疗的疾病类型、给药途径、病人年龄、性别、体重和疾病的严重程度以及作为活性成分的药物类型。根据本发明的一些实施例，日剂量可分为适宜形式的1剂、2剂或多剂，以在整个时间段内以1次、2次或多次给药，只要达到治疗有效量即可。
提高基质金属蛋白酶7活性的方法
在本发明的另一方面，本发明提出了一种提高基质金属蛋白酶7活性的方法。根据本发明的实施例，所述方法包括：将基质金属蛋白酶7与瓜氨酸或其药学上可接受的盐接触。通过将瓜氨酸或其药学上可接受的盐与基质金属蛋白酶7接触，可以提高基质金属蛋白酶7活性，从而有助于基质金属蛋白酶7的科学研究或临床应用。
上调编码基质金属蛋白酶7的基因表达量的方法
在本发明的又一方面，本发明提出了一种上调编码基质金属蛋白酶7的基因表达量的方法。根据本发明的实施例，所述方法包括：将瓜氨酸或其药学上可接受的盐与含有编码基质金属蛋白酶7的基因的细胞或者组织共培养。在适于细胞或组织培养的条件下，瓜氨酸可以有效地提高编码基质金属蛋白酶7的基因表达量，从而有助于基质金属蛋白酶7及其编码基因的科学研究和临床应用。
用于提高免疫力或者预防或治疗焦虑症的药物组合物
在本发明的又一方面，本发明提出了一种用于提高免疫力或者预防或治疗焦虑症的药物组合物。根据本发明的实施例，所述药物组合物包含瓜氨酸或其药学上可接受的盐。
根据本发明的实施例，所述药物组合物进一步包括：药学上可接受的辅料。
本发明对于辅料的种类不做严格限定，可以根据情况灵活选择。对于注射制剂，药物上可接受的载体可以包括缓冲剂、防腐剂、止痛剂、增溶剂等渗压剂(isotonicagent)和稳定剂。对于局部给药的制剂，药物上可接受的载体可以包括碱、赋形剂、润滑剂和防腐剂。本发明的药物组合物可以与上述的药物上可接受的载体结合被制备成各种剂型。对于注射制剂，药物组合物可以被制备成例如一次剂量的剂型的安瓿或例如多剂量容器的单元型剂 型。药物组合物还可以被制备成溶液、悬浮液、药片、药丸、胶囊和长效制剂。其中，根据本发明的一些具体示例，适合药物配方的辅料中赋形剂和稀释液的可以包括：乳糖、葡萄糖、蔗糖、山梨糖醇、甘露醇、木糖醇、赤藻糖醇、麦芽糖醇、淀粉、阿拉伯橡胶、藻酸盐、凝胶、磷酸钙、硅酸钙、纤维素、甲基纤维素、微晶纤维素、聚乙烯吡咯烷酮、水、羟基苯甲酸甲酯、羟苯丙酯、滑石、硬脂酸镁和矿物油。根据本发明的另一些实施例，本发明的辅料还可以包括填充剂、抗凝血剂、润滑剂、保湿剂、芳香剂和防腐剂。
预防或治疗焦虑症或提高免疫力的方法
在本发明的又一方面，本发明提出了一种预防或治疗焦虑症或提高免疫力的方法。根据本发明的实施例，所述方法包括：向受试者施用瓜氨酸或其药学上可接受的盐。
本发明对于受试者不做严格限定，可以为人或其他动物，例如牛、马、鼠、兔、猪等。
根据本发明的实施例，所述施用的方式为口服或者灌肠。
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1
1、选取8月龄的野生小鼠和mtDNA突变小鼠，通过免疫组化实验检测肠道MMP7的表达，免疫组化实验步骤如下：
(1)取材。取约3-5cm小肠中肠肠段，使用冷PBS溶液对肠段中的食糜进行冲洗，注意冲洗力度，避免破坏小肠结构。随后将肠段完全浸泡于4％多聚甲醛中，固定2天。
(2)脱蜡与水化。依次对切片进行以下处理：二甲苯浸泡两次，每次更换新的二甲苯，每次五分钟；100％酒精浸泡两次，每次更换新的100％酒精，每次3分钟；90％酒精浸泡3分钟；80％酒精浸泡3分钟；70％酒精浸泡3分钟；蒸馏水浸泡1分钟；磷酸缓冲盐溶液(PBS)浸泡三次，每次5分钟。
(3)抗原微波修复。使用PBS将EDTA抗原修复液(50×,pH9)稀释成1×，将切片浸泡在1×EDTA抗原修复液中，依次使用微波炉100火力高档热10分钟，50火力低档热10分钟后，取出玻片，使其自然冷却。冷却后，将切片置于PBS溶液中浸泡三次。每次5分钟。在组织块周围使用组化笔画上疏水圈。接下来步骤将使用伊莱瑞特公司的二步法免疫组化试剂盒(货号：E-IR-R217-18mL)进行。在疏水圈中滴加50微升E-IR-R217C(3％ H2O2)试剂，室温10分钟以灭活内源性酶。将切片浸泡在PBS溶液中，浸泡三次，每次更换新的PBS溶液，每次3分钟。在疏水圈中滴加E-IR-R217A(正常山羊血清)，37℃封闭30分钟。封闭结束后，甩去多余液体。在疏水圈中滴加使用E-IR-R217A(正常山羊血清)稀释的MMP7抗体(货号：3801S)(1:100稀释)，4℃孵育12小时。将切片浸泡在PBS溶液中，浸泡三次，每次更换新的PBS溶液，每次3分钟。在疏水圈中滴加E-IR-R217B(Polyperoxidase-anti-Mouse/Rabbit IgG)，37℃孵育30分钟，将切片浸泡在PBS溶液中，浸泡三次，每次更换新的PBS溶液，每次3分钟。每1mL E-IR-R217E(DAB底物液)中，加50μL的E-IR-R217D(DAB浓缩液)，混合均匀为DAB工作液，在疏水圈中滴加50μL DAB工作液，室温避光孵育5分钟。用自来水洗5分钟以终止显色。
(4)苏木素复染、分化、返蓝。将切片放置在苏木素染料中浸泡30秒，自来水冲洗2分钟。将切片放置在1％盐酸酒精染料中浸泡5秒，随后将切片放置稀碳酸锂水溶液2分钟。自来水冲洗3分钟。
(5)脱水与透明。依次在70％、80％、90％、100％(两次)酒精中各浸泡3分钟，二甲苯(两次)依次浸泡3分钟和2分钟。
(6)中性树脂封片。
(7)使用3D HISTECH Pannoramic 250FLASH扫片，3D HISTECH CaseViewer进行阅片。
结果如图1所示，mtDNA突变小鼠MMP7表达量下降，肠道菌群失调，抗菌免疫能力反应减弱。
2、选取8月龄的野生小鼠和mtDNA突变小鼠转移至旷场实验的实验室，目的让小鼠适应新的环境。适应一天后，小鼠即进行旷场实验。首先将小鼠置于50×50×50cm(长×宽×高)的小室中央10分钟，用位于方盒上方的摄像机进行记录，用Noulds EthoVision 9.0软件进行操作。在两次测试之间，用75％的酒精清洗场地。进行旷场实验期间，记录并分析进入中心区的平均速度和频率等所有行为，评估8月龄野生型和mtDNA突变小鼠的焦虑水平。
如图2所示，旷场实验结果mtDNA小鼠进入中央区的频率和平均速度较少，结果表明mtDNA突变小鼠的焦虑增加。这些结果表明了焦虑过程脑肠轴的功能受到了影响。
3、为了寻求调控脑-肠轴功能的潜在机制，检测mtDNA突变小鼠的小肠代谢物组学，具体步骤如下：
(1)取小肠，取约3-5cm小肠中肠肠段，使用冷PBS溶液对肠段中的食糜进行冲洗，注意冲洗力度，避免破坏小肠结构，立刻液氮速冻。样本从-80℃冰箱中取出，冰上解冻，解冻后称取样本50mg于2mL EP管中，加入500uL-20℃预冷的70％甲醇水内标提取液。
(2)加入一颗小钢珠，30Hz匀浆4次，每次30s。
(3)匀浆完后1500r/min振荡5min，冰上静置15min。
(4)离心10min，4℃，12000r/min。
(5)取上清200uL装到对应进样瓶内衬管中，用于LC-MS/MS分析。
结果如图3所示，mtDNA突变小鼠中小肠瓜氨酸特异性下降，提示瓜氨酸在脑-肠轴中发挥重要作用。
实施例2
称量200mg L型瓜氨酸并将其加入到250mLSPF级动物饮用水，充分混匀溶解。对8月龄mtDNA突变小鼠进行体重称量，并将小鼠按体重平均分为两组，一组为250mLSPF级动物饮用水(对照组)，另一组为含有200mg L型瓜氨酸的250mLSPF级动物饮用水(实验组)。14天无间断给水。经过14天无间断给水后，通过免疫组化实验检测小肠的MMP水平、通过旷场实验检测小鼠的焦虑水平。。
结果如图4和图5所示，瓜氨酸给药后增加了mtDNA突变小鼠的MMP7表达量，有助于提高免疫力。并且，旷场实验结果表达加入瓜氨酸后，mtDNA突变小鼠进入中央区的频率和平均速度增加，表明焦虑较少，说明瓜氨酸缓解了mtDNA突变小鼠的焦虑。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (11)

  1. 瓜氨酸或其药学上可接受的盐在制备药物中的用途，其特征在于，所述药物用于预防或治疗焦虑症或提高免疫力。
  2. 根据权利要求1所述的用途，其特征在于，所述药物用于预防或治疗肠道菌群失调。
  3. 根据权利要求1所述的用途，其特征在于，所述药物用于提高基质金属蛋白酶7活性或者上调编码基质金属蛋白酶7的基因表达量。
  4. 根据权利要求1所述的用途，其特征在于，所述药物靶向脑-肠轴。
  5. 根据权利要求1所述的用途，其特征在于，所述药物的给药方式为口服或者灌肠。
  6. 一种提高基质金属蛋白酶7活性的方法，其特征在于，包括：将基质金属蛋白酶7与瓜氨酸或其药学上可接受的盐接触。
  7. 一种上调编码基质金属蛋白酶7的基因表达量的方法，其特征在于，包括：
    将瓜氨酸或其药学上可接受的盐与含有编码基质金属蛋白酶7的基因的细胞或者组织共培养。
  8. 一种用于提高免疫力或者预防或治疗焦虑症的药物组合物，其特征在于，所述药物组合物包含瓜氨酸或其药学上可接受的盐。
  9. 根据权利要求8所述的药物组合物，其特征在于，进一步包括：药学上可接受的辅料。
  10. 一种预防或治疗焦虑症或提高免疫力的方法，其特征在于，包括：向受试者施用瓜氨酸或其药学上可接受的盐。
  11. 根据权利要求10所述的方法，其特征在于，所述施用的方式为口服或者灌肠。
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