WO2024061169A1 - 普雷沃氏菌在防治注意缺陷多动障碍中的应用 - Google Patents

普雷沃氏菌在防治注意缺陷多动障碍中的应用 Download PDF

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WO2024061169A1
WO2024061169A1 PCT/CN2023/119447 CN2023119447W WO2024061169A1 WO 2024061169 A1 WO2024061169 A1 WO 2024061169A1 CN 2023119447 W CN2023119447 W CN 2023119447W WO 2024061169 A1 WO2024061169 A1 WO 2024061169A1
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prevotella
attention deficit
hyperactivity disorder
deficit hyperactivity
rats
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PCT/CN2023/119447
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English (en)
French (fr)
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邱景富
陈承志
邹镇
刘一鋆
郭长新
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重庆医科大学
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Publication of WO2024061169A1 publication Critical patent/WO2024061169A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • 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
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia

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  • the invention belongs to the field of biomedicine technology, and specifically relates to the application of Prevotella in preventing and treating attention deficit hyperactivity disorder.
  • the human intestine provides a good habitat for microorganisms.
  • the number of microorganisms colonizing the intestine is about 10 trillion, which is 10 times the number of human cells.
  • the genes encoded by them are also 150 times that of the human genome.
  • intestinal microorganisms themselves and their metabolites can not only regulate human health, but also play an important role in bridging the diet and the host. They are a very important component of the human health system. .
  • Intestinal microorganisms can regulate the central nervous system through the microbiome-gut-brain axis and influence the occurrence and development of neurobehavioral disorders.
  • ADHD Attention-Deficit/Hyperactivity Disorder
  • gut immune dysfunction is part of the pathophysiology of ADHD.
  • atopic diseases such as allergic rhinitis, asthma, allergic dermatitis, and allergic conjunctivitis has been increased in hospitalized patients with ADHD.
  • the incidence of constipation and fecal incontinence has been increased in children with ADHD.
  • gut dysbiosis may be associated with gastrointestinal dysfunction in patients with ADHD and may play a role in brain function and behavior in these cases.
  • the intestinal flora of children with ADHD is significantly different in diversity.
  • the discovery of the "microbiome-gut-brain axis" confirms that the intestinal flora is closely related to the nervous system, and intestinal microorganisms can pass through Affects multiple neurotransmitter, immune, endocrine, and metabolic pathways to affect the central nervous system, resulting in behavioral and cognitive changes. Rebuilding the homeostasis of intestinal flora is a new means to prevent and alleviate diseases related to mental and behavioral abnormalities.
  • ADHD has been mainly treated with monoaminergic psychostimulants, mainly methylphenidate (MPH) and amphetamines (AMPH), plus the catecholaminergic non-stimulant atomoxetine (ATX).
  • MPH methylphenidate
  • AMPH amphetamines
  • ATX catecholaminergic non-stimulant atomoxetine
  • MPH methylphenidate
  • AMPH amphetamines
  • ATX catecholaminergic non-stimulant atomoxetine
  • MPH methylphenidate
  • AMPH amphetamines
  • ATX catecholaminergic non-stimulant atomoxetine
  • Prevotella belongs to the phylum Bacteroidetes and is a Gram-negative anaerobic bacterium. Prevotella is often considered a bacterium associated with a healthy plant-based diet, functioning as a "probiotic" in the human body. Reductions in the genus Prevotella are associated with certain diseases. Prevotella genus abundance was lower in children diagnosed with autism. Decreased levels of Prevotella in infants are associated with behavioral problems at two years of age. Some dietary studies have reported that Prevotella is positively related to a diet rich in polysaccharides and fiber. In addition, the abundance of Prevotella was positively correlated with the weekly exercise time of cyclists; in the elderly with constipation, the abundance of Prevotella was reduced.
  • the present invention aims to solve one of the technical problems in the related art, at least to a certain extent.
  • the main purpose of the present invention is to provide an application of Prevotella in preventing and treating attention deficit hyperactivity disorder.
  • the Prevotella is used to treat attention deficit hyperactivity disorder.
  • the Prevotella is used in a therapeutically effective amount to achieve attention deficit hyperactivity disorder.
  • the attention deficit hyperactivity disorder includes, but is not limited to, attention deficit hyperactivity disorder caused by abnormal brain functional development.
  • the effective dose of Prevotella is 200 ⁇ L, and the concentration of the Prevotella liquid is 2.8 ⁇ 10 8 CFU/mL.
  • the Prevotella bacteria are formulated into pharmaceutical preparations, and the pharmaceutical preparations include any one of liquid preparations, solid preparations, semi-solid preparations and gas preparations.
  • the dosage form of the pharmaceutical preparation includes any pharmaceutically acceptable dosage form.
  • the pharmaceutical preparation also includes any one or more of pharmaceutically acceptable pharmaceutical excipients.
  • the administration method of Prevotella is intragastric administration.
  • the present invention at least has the following advantages:
  • the present invention provides a new use of Prevotella, which is used to prevent and treat attention deficit hyperactivity disorder. It is a probiotic strain that relieves abnormal psychological and behavioral states related to ADHD by balancing intestinal microorganisms and regulating endocrine dysfunction of the brain-gut axis. It has been verified through animal experiments that Prevotella can significantly improve hyperactive behavior, spontaneous activity levels and impulsive behavior. Further experiments have found that Prevotella can significantly increase the levels of adrenocorticotropic hormone and adrenocorticotropic hormone in the serum.
  • Cortisol levels improve neuroendocrine system dysfunction, and significantly increase glucocorticoid receptor (Nr3c1) and mineralocorticoid receptor (Nr3c2) gene expression, thereby alleviating ADHD social dysfunction, providing prospects for the application of probiotics in relieving ADHD. theoretical basis.
  • Figure 1 is the growth curve of Prevotella in the present invention.
  • Figure 2 shows the concentration of Prevotella bacteria liquid in the present invention
  • FIG 3 shows the effect of Prevotella in the present invention on the hyperactive behavior and spontaneous activity level of attention deficit hyperactivity disorder rats (SHR);
  • Figure 4 shows the effect of Prevotella on the impulsive behavior of attention deficit hyperactivity disorder rats (SHR) in the present invention
  • Figure 5 shows the effect of Prevotella in the present invention on hormones related to the neuroendocrine system of the midgut-brain axis in the brain tissue of attention deficit hyperactivity disorder rats (SHR);
  • Figure 6 shows the effect of Prevotella in the present invention on the expression of genes related to the neuroendocrine system of the midgut-brain axis in the brain tissue of attention deficit hyperactivity disorder rats (SHR).
  • SHR attention deficit hyperactivity disorder rats
  • Prevotella activation was performed in an anaerobic laboratory. Prepare 1 tube containing 15 mL of thioglycolate fluid medium (15.0 g of caseeptone (tryptic hydrolysis), 5.0 g of yeast extract powder, 5.0 g of glucose, 0.5 g of sodium thioglycolate, 0.5 g of L-cystine, chlorinated Sodium 2.5g, resazurin 0.001g, agar 0.75g, pH value 7.1 ⁇ 0.2) test tube (placed in an anaerobic environment for 24 hours in advance to remove oxygen); disinfect the surface of the purchased ampoule tube containing the freeze-dried powder of Prevotella strain with alcohol, and burn the Prevotella strain with an alcohol lamp.
  • thioglycolate fluid medium 15.0 g of caseeptone (tryptic hydrolysis), 5.0 g of yeast extract powder, 5.0 g of glucose, 0.5 g of sodium thioglycolate, 0.5 g of L-cy
  • the top of the strain ampoule tube is quickly moved to an anaerobic incubator to cool and rupture.
  • the turbidimetric method was used.
  • the growth curve of Prevotella is shown in FIG1 , which is the growth curve of Prevotella.
  • the number of live bacteria of Prevotella increases rapidly within 60h to 72h.
  • Prevotella has adapted to the culture medium environment, the bacterial colony is vigorous, and it begins to grow and reproduce at a high speed, and is in the logarithmic growth phase.
  • the concentration of Prevotella bacterial solution is shown in Figure 2, where A is the colony growth of Prevotella original solution after culturing for 72 hours on Columbia blood agar petri dishes. B is the colony growth of Prevotella bacteria solution diluted 10 -6 and cultured on Columbia blood agar petri dishes for 72 hours. C is the colony growth of Prevotella bacteria solution diluted 10 -7 and cultured on Columbia blood agar petri dishes for 72 hours. D is the colony growth of Prevotella bacteria solution diluted 10 -8 and cultured on Columbia blood agar petri dishes for 72 hours.
  • Prevotella reaches the logarithmic phase 72-80 hours after culture.
  • Use a pipette to draw 1 mL of bacterial liquid from the logarithmic growth phase culture into the spectrophotometer sample pool, and use a UV spectrophotometer to detect the OD 600 value. (Optical Density) and determine the concentration of the bacterial solution. When OD 600 ⁇ 1, the concentration of bacterial solution is about 10 8 -10 9 cfu/mL.
  • the bacterial stock solution was centrifuged at 6000 ⁇ g for 5 minutes, the supernatant was removed and the pellet was resuspended in sterile PBS to prepare a bacterial suspension of approximately 10 8 -10 9 cfu/mL.
  • the diluted bacterial solution sample is placed in a sterilized centrifuge tube and sealed, and the sample is administered to the experimental animal by gavage in accordance with the requirements for aseptic delivery.
  • the experimental animals selected were: 16 three-week-old SHR male rats were selected, the room temperature was maintained at 23 ⁇ 1°C, and the humidity was maintained at 50 ⁇ 60%. There is a 12-hour light-dark cycle, and clean food and water are available ad libitum for training.
  • SHR rats were divided into Vehicle (SHR) and Prevotella gavage group (P.spp), with 8 rats in each group.
  • Vehicle was given 200 ⁇ L of normal saline by gavage every day, and the P.spp group was given fermentation liquid (viable bacterial count: 10 8 CFU/mL) by gavage.
  • 200 ⁇ L (2*10 8 -2*10 9 cfu) bacterial solution was administered daily.
  • the daily administration time was from 8:00 to 9:00 in the morning for 14 consecutive days. On the 15th day, rats in each group were fasted for 12 hours for behavioral experiments.
  • the open field experiment is used to evaluate rats' autonomous activities, active exploration and anxiety state in a new environment.
  • the open-field device is a black box with an open upper part 50cm long, 50cm wide and 40cm high.
  • Figure 3 shows the effect of Prevotella on the hyperactive behavior and spontaneous activity level of attention deficit hyperactivity disorder rats (SHR).
  • SHR attention deficit hyperactivity disorder rats
  • 3D shows the activity trajectory of Vehicle rats in the open field experiment.
  • 3E is the activity trajectory diagram of rats in the P.spp group in the open field experiment.
  • the elevated plus maze is a vertical cross composed of two closed walls (50cm ⁇ 10cm), two open walls (50cm ⁇ 10cm) and a central area (10cm ⁇ 10cm), 50cm above the ground.
  • the animals were placed in a soundproof room to acclimate to the environment. Strictly control room temperature and ventilation.
  • the experimenter placed the animal in the central area at the same position each time, with the animal's head facing the direction of one of the open walls.
  • the experimenter started the experiment after leaving the experimental device area.
  • the experiment lasted for 5 minutes.
  • the animal's impulsive behavior was evaluated based on the time the animal stayed in the open arm and the percentage of the number of times the animal entered the open arm to the total number of times and the total time.
  • Figure 4 shows the effect of Prevotella on the impulsive behavior of attention deficit hyperactivity disorder rats (SHR).
  • SHR attention deficit hyperactivity disorder rats
  • 4A shows that after two weeks of continuous gavage of Prevotella, the total moving distance of rats in the P.spp group in the elevated plus maze test was significantly reduced
  • 4B shows that after two weeks of continuous gavage of Prevotella, P. The number of times rats in the spp group entered the open arm space was significantly reduced
  • 4C means that after two weeks of continuous administration of Prevotella bacteria, the percentage of the rats in the P.spp group's activity time in the open arm space was significantly reduced in the elevated plus maze.
  • 4D is the activity trajectory of Vehicle rats in the elevated plus maze.
  • 4E is the activity trajectory diagram of rats in the P.spp group in the elevated plus maze.
  • ELISA enzyme-linked immunosorbent was used to determine serum levels of hormones related to the gut-brain axis neuroendocrine system: adrenocorticotropic hormone (ACTH) and cortisol (CORT).
  • ACTH adrenocorticotropic hormone
  • CORT cortisol
  • Quantitative-PCR was used to measure the expression levels of genes related to the neuroendocrine system in the gut-brain axis of the brain tissue from the brain tissues collected in the above animal experiments: glucocorticoid receptor (Nr3c1) and mineralocorticoid receptor (Nr3c2).
  • Prevotella may alleviate ADHD symptoms by regulating HPA axis function.
  • the present invention shows that Prevotella can alleviate ADHD-related hyperactivity and impulsive behavioral symptoms. This effect may be exerted by regulating hormone levels and gene expression levels related to neuroendocrine functions in the gut-brain axis.
  • the present invention provides a strong theoretical basis for seeking the therapeutic potential of probiotics for attention deficit hyperactivity disorder, and has high social significance and potential market value.

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Abstract

提供了一种普雷沃氏菌在防治注意缺陷多动障碍中的应用，该普雷沃氏菌以治疗有效量使用以实现注意缺陷多动障碍的治疗，该普雷沃氏菌的菌株编号为JCM 13464＝DSM 18205。普雷沃氏菌对多动行为、自发性活动水平及冲动行为都有相当显著的改善；普雷沃氏菌能显著提高血清中促肾上腺皮质激素及皮质醇水平，改善神经内分泌系统功能异常，以及显著提高糖皮质激素受体(Nr3c1)，盐皮质激素受体(Nr3c2)基因表达，进而缓解ADHD社会功能障碍。

Description

普雷沃氏菌在防治注意缺陷多动障碍中的应用 技术领域
本发明属于生物医药技术领域，具体涉及一种普雷沃氏菌在防治注意缺陷多动障碍中的应用。
背景技术
人体肠道为微生物提供了良好的栖息环境，在肠道内定植微生物数量约为10万亿个，是人体细胞数的10倍，由其编码的基因也是人类基因组的150倍。作为人体最庞大、最复杂的微生态系统，肠道微生物本身及其代谢产物不仅能调节人体健康，更在膳食和宿主之间起到了重要的桥梁作用，是人体健康系统中非常重要的组成部分。肠道微生物可以通过微生物-肠-脑轴对中枢神经系统进行调控，影响神经性行为障碍疾病的发生发展。
注意缺陷多动障碍(Attention-Deficit/Hyperactivity Disorder，ADHD)是儿童最常见的神经发育性障碍之一，核心症状为注意缺陷、多动/冲动，伴有社交、学业等方面功能损害。全世界儿童ADHD的患病率约为7.2％，我国儿童ADHD患病率为6.26％。由于其症状多在学龄期出现，因此数十年来ADHD被认为是一种局限于儿童、青少年的行为问题。但是到20世纪80年代初，相继有许多学者对ADHD进行了追踪研究，结果发现10％-60％的ADHD患者到青春期后，其症状尤其是社会功能并未缓解，只是ADHD的临床表现形式发生了变化，而且出现了更多的共患病和社会问题，对患者的学业、家庭和社会生活等方面产生了明显不良的影响。因此，ADHD已经越来越引起人们更广泛地关注。
有证据表明，肠道免疫功能障碍是ADHD病理生理学的一部分。过敏性鼻炎、哮喘、过敏性皮炎和过敏性结膜炎等特应性疾病的发病率在住院ADHD患者中有所增加。在患有多动症的儿童中，便秘和大便失禁的发生率都在增加[24]。这些发现表明，肠道生态失调可能与ADHD患者的胃肠功能障碍有关，并可能在这些病例中对大脑功能和行为起作用。
ADHD患儿的肠道菌群与正常儿童相比，多样性上有显著差异，此外“微生物-肠-脑轴”的发现，证实了肠道菌群与神经系统关系密切，肠道微生物可通过影响多种神经递质、免疫、内分泌和代谢途径来影响中枢神经系统，进而导致行为和认知变化。重建肠道菌群稳态，是预防和缓解精神行为异常性相关疾病的新手段。
70多年来，多动症的治疗主要是使用单胺能精神刺激药，主要是哌甲酯(MPH)和安非他明(AMPH)，外加儿茶胺能非兴奋剂阿托西汀(ATX)。然而在临床上大约有20％-30％的患者使用MPH治疗效果差甚至无效，不能达到症状的 控制。还有一部分患者因不能耐受MPH的副作用(睡眠障碍、食欲降低、头痛、抽搐等)而减少药量甚至停药，也不能达到临床控制。另外由于中枢兴奋药物远期疗效的局限性和潜在的滥用危险，一些家庭拒绝中枢兴奋药物的治疗。因此，寻找副作用小、价格经济且适合预防敏感人群ADHD发生的产品，非常的必要和紧迫。
普雷沃氏菌属于拟杆菌门，是革兰氏阴性厌氧菌。普雷沃氏菌通常被认为是一种与健康的植物性饮食相关的菌，在人体中发挥“益生菌”的作用。普雷沃氏菌属的减少与某些疾病相关。被诊断患有自闭症的儿童的普雷沃氏菌属丰度较低。婴儿普雷沃氏菌水平的下降与两岁时的行为问题有关。曾有一些饮食方面的研究报道，Prevotella菌和富含多糖和纤维的饮食正相关。此外，Prevotella的丰度还和自行车运动员每周的运动时间正相关；在便秘的老人中，Prevotella菌的丰度降低。然而，目前尚未有普雷沃氏菌能否改善注意缺陷多动障碍症状的研究报道。当前市场上的益生菌产品，多仅以补充益生菌为目标，没有深入研究益生菌株的功能，远远低估了益生菌的作用，更没有与ADHD相关的益生菌产品。
发明内容
本发明旨在至少在一定程度上解决相关技术中的技术问题之一。为此，本发明的主要目的在于提供一种普雷沃氏菌在防治注意缺陷多动障碍中的应用。
本发明的目的是通过以下技术方案实现的：
一种普雷沃氏菌在防治注意缺陷多动障碍中的应用，所述普雷沃氏菌用于治疗注意缺陷多动障碍，所述普雷沃氏菌以治疗有效量使用以实现注意缺陷多动障碍的治疗，该普雷沃氏菌的菌株编号为JCM 13464＝DSM 18205。
进一步，所述注意缺陷多动障碍包括但不限于大脑功能发育异常引起的注意缺陷多动障碍。
在某些具体实施方式中，所述普雷沃氏菌的有效剂量为200μL，且所述普雷沃氏菌菌液的浓度为2.8×108CFU/mL。
在某些具体实施方式中，所述普雷沃氏菌制成药物制剂，所述药物制剂包括液体制剂、固体制剂、半固体制剂和气体制剂中的任一种。
在某些具体实施方式中，所述药物制剂的剂型包括药剂学上可接受的任意一种剂型。
进一步，所述药物制剂还包括药剂学上可接受药用辅料中的任一种或者多种。
在某些具体实施方式中，所述普雷沃氏菌的给药方式为灌胃给药。
与现有技术相比，本发明至少具有以下优点：
1)本发明提供了该普雷沃氏菌的一种新用途，其用于防治注意缺陷多动 障碍的相关症状，其是通过平衡肠道微生物、调节脑肠轴内分泌功能紊乱，从而缓解与ADHD相关的异常心理行为状态的益生菌株。通过动物实验验证，普雷沃氏菌对多动行为、自发性活动水平及冲动行为都有相当显著的改善；且通过实验进一步探索发现普雷沃氏菌能显著提高血清中促肾上腺皮质激素及皮质醇水平，改善神经内分泌系统功能异常，以及显著提高糖皮质激素受体(Nr3c1)，盐皮质激素受体(Nr3c2)基因表达，进而缓解ADHD社会功能障碍，为益生菌缓解ADHD的应用前景提供了理论依据。
附图说明
为了更清楚地说明本发明具体实施方式，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍。
图1为本发明中普雷沃氏菌的生长曲线；
图2为本发明中普雷沃氏菌菌液浓度；
图3为本发明中普雷沃氏菌对注意缺陷多动障碍大鼠(SHR)的多动行为、自发性活动水平的影响；
图4为本发明中普雷沃氏菌对注意缺陷多动障碍大鼠(SHR)的冲动行为的影响；
图5为本发明中普雷沃氏菌对注意缺陷多动障碍大鼠(SHR)脑组织中肠脑轴神经内分泌系统相关激素的影响；
图6为本发明中普雷沃氏菌对注意缺陷多动障碍大鼠(SHR)脑组织中肠脑轴神经内分泌系统相关基因表达的影响
具体实施方式
为了使本发明实现的技术手段、创作特征、达成目的与功效易于明白了解，下面结合具体实施例和附图，进一步阐述本发明，但下述实施例仅仅为本发明的优选实施例，并非全部。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径获得。
本申请中的普雷沃氏菌，保藏于上海保藏生物技术中心，保藏机构简称：SHBCC，菌株编号：JCM 13464＝DSM 18205。
实施例1普雷沃氏菌的培育
本申请中的普雷沃氏菌，通过下述培育方法获得，具体包括如下步骤：
①普雷沃氏菌活化
普雷沃氏菌活化操作在厌氧操作台中进行。准备1支含有15mL硫乙醇酸盐流体培养基(酪胨(胰酶水解)15.0g，酵母浸出粉5.0g，葡萄糖5.0g，硫乙醇酸钠0.5g，L-胱氨酸0.5g，氯化钠2.5g，刃天青0.001g，琼脂0.75g， pH值7.1±0.2)的试管(提前置于厌氧环境除氧24h)；将购买的装有普雷沃氏菌株冻干粉的安瓿管表面酒精消毒后，用酒精灯灼烧普雷沃氏菌株安瓿管顶部后迅速移至厌氧培养箱中冷却使之破裂。将0.5mL硫乙醇酸盐流体培养基加入安瓿管中，充分溶解冻干粉后，将普雷沃氏菌株混悬液加入到含有硫乙醇酸盐流体培养基的试管中，封口后放于15×30cm的密封厌氧培养袋，并立刻放入厌氧产气袋，在37℃厌氧培养箱(湿度65％)中静置培养至菌液浑浊或底部有大量菌体生长。
②普雷沃氏菌生长曲线的测定
为了测定普雷沃氏菌的数量，了解生长状况和代谢情况，采用比浊法测定。利用分光光度计测定普雷沃氏菌悬液与OD值的关系，以此明确普雷沃氏菌生长规律，并将其与所培养时间对应，明确普雷沃氏菌生长曲线，为实现后续体内外实验菌液的均一性准备。
其中普雷沃氏菌生长曲线如图1所示，图1为普雷沃氏菌的生长曲线。从图中可知，普雷沃氏菌的活菌数在60h至72h内迅速升高。此时普雷沃氏菌已适应了培养基环境，菌群活力旺盛，开始高速生长繁殖，处于生长对数期。
③普雷沃氏菌菌液浓度计算
吸取0.1mL混合液于盛有10mL硫乙醇盐酸盐液体培养基的试管中，此稀释度为10-1，重复以上过程作10倍比稀释，至10-8稀释度，选择10-6，10-7，10-8三个稀释度，吸取0.1mL菌液滴于哥伦比亚血琼脂培养基(胰酪蛋白胨5.0g，大豆蛋白胨5.0g，酵母浸粉10.0g，葡萄糖10.0g，L-半胱氨酸0.5g，刃天青0.001g，磷酸二氢钾0.04g，磷酸氢二钾0.04g，碳酸氢钠0.4g，氯化钠0.08g，氯化钙0.008g，硫酸镁0.0192g，5％无菌脱纤维羊血，多粘菌素B 0.001g，pH值7.0)平板上，平板涂布后采用厌氧培养法(5％CO2)将涂好的培养皿置37℃培养箱过夜培养。平板过夜长至菌落清晰不想连(12-24h)，数菌落个数(n)。根据计数结果绘制标准曲线，计算菌浓度。
计算公式：N(cfu/mL)＝n*d2/V
n:平均菌落数d2:稀释浓度v:涂板菌液体积
其中普雷沃氏菌菌液浓度如图2所示，其中A为普雷沃氏菌原液在哥伦比亚血琼脂培养皿上培养72h后的菌落生长情况。B为普雷沃氏菌菌液稀释10-6在哥伦比亚血琼脂培养皿上培养72h后的菌落生长情况。C为普雷沃氏菌菌液稀释10-7在哥伦比亚血琼脂培养皿上培养72h后的菌落生长情况。D为普雷沃氏菌菌液稀释10-8在哥伦比亚血琼脂培养皿上培养72h后的菌落生长情况。
④普雷沃氏菌传代培养与保存
准备10支装有硫乙醇酸盐流体培养基试管，取500μL活化好的普雷沃氏 菌液接种于试管中，封口后放于15×30cm的密封厌氧培养袋，并放入厌氧产气袋。在37℃厌氧箱中静置培养1天后，明显可见液体培养基中菌液浑浊。菌种冻存管和60％无菌甘油(体积分数)高压灭菌后放入厌氧培养箱中，然后取菌液和甘油按照1：1的比例移入冻存管中，每管中加入1mL左右的细菌悬液，使甘油终浓度达到30％，-80℃保存。
⑤普雷沃氏菌菌液配置
普雷沃氏菌在培养后的72-80h达到对数期，用移液枪从对数生长期培养物中吸取1mL菌液于分光光度计样品池中，用紫外分光光度计检测OD600值(Optical Density)并确定菌液浓度。当OD600≈1时，菌液浓度约108-109cfu/mL。细菌原液以6000×g离心5分钟后，去除上清液并将沉淀重悬于无菌PBS中制成约108-109cfu/mL细菌悬液。将该稀释菌液样本分装于灭菌离心管内密封，按照无菌传递要求将该样品对实验动物进行灌胃。
实施例2普雷沃氏菌的效果验证和探索实验
下述实验中，所选用的实验动物为：选取16只三周龄SHR雄性大鼠，室温保持在23±1℃，湿度保持在50±60％。光暗循环12小时，可随意获取清洁食品和水进行训化。
实验方法：SHR大鼠分为Vehicle(SHR)、普雷沃氏菌灌胃组(P.spp)，每组8只。Vehicle每天灌胃给予200μL生理盐水，P.spp组灌胃发酵液(活菌数108CFU/mL)。每日灌胃给予200μL(2*108-2*109cfu)菌溶液。每日给药时间为上午8：00－9:00，连续给予14d，第15天各组大鼠空腹12h，进行行为学实验。第16天，各组大鼠空腹12h，眼眶取血，3000r/min离心，保留血清备用。取脑组织(皮层、海马)用于Elisa试剂盒、Q-PCR法等检测。
实验数据用平均值±标准差(SEM)表示，采用独立样本T检验评估两组间差异，所有的图表都是由GraphPadPrism 8.0创建。
1)普雷沃氏菌对多动行为、自发性活动水平的影响
为了测试本申请所提供的普雷沃氏菌对多动行为、自发性活动水平以及冲动行为的影响，本申请将进行如下测试：
①旷场实验
旷场实验是用来评估大鼠在新环境中的自主活动，主动探索和焦虑状态。旷场装置为长50cm，宽50cm，高40厘米上部敞开的黑色箱子。旷场装置中正上方有摄像机与电脑相连，记录大鼠在旷场中的活动信息。实验开始前，在电脑中设置好旷场实验系统参数，并让大鼠适应周围环境5min，然后将大鼠放入旷场中，记录大鼠在旷场内5min的活动情况。每只大鼠实验结束后，对矿场实验装置中的大鼠粪便及尿液进行清理，然后用5％的乙酸水溶液对矿场装置彻底清洁，再用纸巾或干布擦干记录的活动信息包括活动总路程，中央活 动距离，中央活动持续时间和活动轨迹图。
结果如图3所示，图3为普雷沃氏菌对注意缺陷多动障碍大鼠(SHR)的多动行为、自发性活动水平的影响。其中3A为连续灌胃普雷沃氏菌两周后，P.spp组大鼠在旷场实验中的总活动距离显著减少；3B为连续灌胃普雷沃氏菌两周后，P.spp组大鼠在旷场实验中的中心区活动距离显著减少；3C为连续灌胃普雷沃氏菌两周后，P.spp组大鼠在旷场实验中的中心区活动时间显著减少。数据表示为平均值±标准差，N＝6，*P<0.05；**P<0.01；***P<0.001。3D为Vehicle大鼠在旷场实验中的活动轨迹图。3E为P.spp组大鼠在在旷场实验中的活动轨迹图。
②高架十字迷宫
高架十字迷宫是由两个闭合壁(50cm×10cm)、两个开放壁(50cm×10cm)和中央区(10cm×10cm)构成的垂直十字，距离地面高50cm。实验开始前2h，将动物放入隔音房间以适应环境。严格控制房间温度和通风。实验者每次于同一位置将动物放于中央区位置，动物头部朝向其中一个开放壁的方向，实验人员离开实验装置区域后开始实验。实验时间5min，以动物在开臂内停留时间和进入开臂次数占总次数和总时间的百分比为主要指标评价动物冲动行为。
结果如图4所示，图4为普雷沃氏菌对注意缺陷多动障碍大鼠(SHR)的冲动行为的影响。其中4A为连续灌胃普雷沃氏菌两周后，P.spp组大鼠在高架十字迷宫实验中的总活动距离显著减少；4B为连续灌胃普雷沃氏菌两周后，P.spp组大鼠在进入开臂空间次数显著减少；4C为连续灌胃普雷沃氏菌两周后，P.spp组大鼠在在高架十字迷宫中开臂空间活动时间的百分比显著减少。数据表示为平均值±标准差，N＝6，*P<0.05；**P<0.01；***P<0.001。4D为Vehicle大鼠在高架十字迷宫中的活动轨迹图。4E为P.spp组大鼠在高架十字迷宫中的活动轨迹图。
2)普雷沃氏菌对脑组织中肠脑轴神经内分泌系统相关激素的影响
对上述动物实验方法中采集的血清，使用ELISA酶联接免疫吸附剂测定血清中肠脑轴神经内分泌系统相关激素水平：促肾上腺皮质激素(ACTH)，皮质醇(CORT)。
结果如图5所示，图5普雷沃氏菌对注意缺陷多动障碍大鼠(SHR)脑组织中肠脑轴神经内分泌系统相关激素的影响。其中5A为相较于Vehicle大鼠，灌胃两周普雷沃氏菌菌液的大鼠血清中促肾上腺皮质激素(ACTH)明显升高。5B为灌胃两周普雷沃氏菌菌液的大鼠血清中皮质醇(CORT)水平显著高于Vehicle大鼠。数据表示为平均值±标准差，N＝6，*P<0.05；**P<0.01；***P<0.001。酶联接免疫吸附剂测定实验结果表明，普雷沃氏菌可改善注意缺陷多动障碍存在的内分泌系统功能紊乱，调节HPA轴功能。
3)普雷沃氏菌对脑组织中肠脑轴神经内分泌系统相关基因表达的影响
对上述动物实验方法中采集的脑组织，使用Quantitative-PCR法测定脑组织中肠脑轴神经内分泌系统相关基因表达水平：糖皮质激素受体(Nr3c1)，盐皮质激素受体(Nr3c2)。
结果如图6所示，其中6A为普雷沃氏菌对注意缺陷多动障碍大鼠(SHR)海马Nr3c1 mRNA表达水平的影响。6B为普雷沃氏菌对注意缺陷多动障碍大鼠(SHR)海马Nr3c2 mRNA表达水平的影响。数据表示为平均值±标准差，N＝6，*P<0.05；**P<0.01；***P<0.001。灌胃两周普雷沃氏菌菌液后，相较于Vehicle组，P.spp组脑组织中的Nr3c1、Nr3c2 mRNA表达水平显著上调。这些结果表明，普雷沃氏菌可能通过一定的作用机制调节脑肠轴神经内分泌系统功能，进而改善ADHD相关症状。
本发明通过在给予SHR大鼠两周普雷沃氏菌菌液灌胃后，其多动行为、自发性活动水平及冲动行为都有相当显著的改善，且HPA轴相关激素、基因表达都明显上调，根据以上实验结果，推测普雷沃氏菌可能通过调节HPA轴功能缓解ADHD症状。
综上所述，本发明表明普雷沃氏菌具有缓解ADHD相关多动及冲动行为症状。这种效应可能是通过调节肠脑轴中神经内分泌功能相关的激素水平、基因表达水平发挥作用的。本发明对于寻求益生菌对注意缺陷多动障碍的治疗潜力方面提供了有力的理论依据，具有较高的社会意义及潜在的市场价值。
以上实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述各实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围，其均应涵盖在本发明的权利要求和说明书的范围当中。

Claims (7)

  1. 一种普雷沃氏菌在防治注意缺陷多动障碍中的应用，其特征在于，所述普雷沃氏菌用于治疗注意缺陷多动障碍，所述普雷沃氏菌以治疗有效量使用以实现注意缺陷多动障碍的治疗，该普雷沃氏菌的菌株编号为JCM 13464＝DSM18205。
  2. 根据权利要求1所述的应用，其特征在于，所述注意缺陷多动障碍包括但不限于大脑功能发育异常引起的注意缺陷多动障碍。
  3. 根据权利要求1所述的应用，其特征在于，所述普雷沃氏菌的有效剂量为200μL，且所述普雷沃氏菌菌液的浓度为2.8×108CFU/mL。
  4. 根据权利要求1所述的应用，其特征在于，所述普雷沃氏菌制成药物制剂，所述药物制剂包括液体制剂、固体制剂、半固体制剂和气体制剂中的任一种。
  5. 根据权利要求4所述的应用，其特征在于，所述药物制剂的剂型包括药剂学上可接受的任意一种剂型。
  6. 根据权利要求4所述的应用，其特征在于，所述药物制剂还包括药剂学上可接受药用辅料中的任一种或者多种。
  7. 根据权利要求1所述的应用，其特征在于，所述普雷沃氏菌的给药方式为灌胃给药。
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