WO2021008149A1 - Akkermansia muciniphila在制备抗抑郁药物或者保健品中的应用 - Google Patents

Akkermansia muciniphila在制备抗抑郁药物或者保健品中的应用 Download PDF

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WO2021008149A1
WO2021008149A1 PCT/CN2020/079604 CN2020079604W WO2021008149A1 WO 2021008149 A1 WO2021008149 A1 WO 2021008149A1 CN 2020079604 W CN2020079604 W CN 2020079604W WO 2021008149 A1 WO2021008149 A1 WO 2021008149A1
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depression
bacteria
preparation
akkermansia muciniphila
preventing
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陈玉根
陈拓
袁晓敏
王琼
施国平
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江苏省中医院
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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
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • 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
    • A61P25/08—Antiepileptics; Anticonvulsants
    • 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
    • A61P25/24—Antidepressants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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 relates to a probiotic, in particular to the new use of Akkermansia Mucinifila in antidepressant drugs or health care products, and belongs to the technical field of medicine.
  • Depression is a psychiatric disease with persistent and significant depression as its main clinical feature. It is often clinically accompanied by depression, low self-esteem, depression, and even suicide attempts or behaviors, and most patients have a tendency to recur. Therefore, depression is a disease with a high disability rate. According to the World Health Organization (WHO), 15 out of every 100 people in the general population have suffered from depression. The annual expenditure for depression treatment accounts for 12.3% of total medical expenditure. In China, there are estimated to be 20-50 million depression patients, and the prevalence is increasing year by year. At present, there is no safe and effective antidepressant biologics in the market.
  • WHO World Health Organization
  • Akkermansiamuciniphila is a bacterial species identified in 2004. It is a common symbiotic bacteria in the human digestive tract, accounting for about 3-5%. Akkermansiamuciniphila has been confirmed to be closely related to chronic diseases such as obesity, type 2 diabetes, and autism. However, it has not been reported in the prevention and treatment of depression.
  • the application of Akkermansia Muciniphila bacteria in the preparation of medicines or health products for preventing and treating intestinal diseases and depression are preferred.
  • the intestinal diseases include enteritis and intestinal tumors.
  • a biological preparation for preventing and treating depression which includes Akkermansia Mucinphila bacteria or Akkermansia Mucin ⁇ phila bacteria extract.
  • the Akkermansia Mucinphila bacteria include live Akkermansia Mucininiphila bacteria or pasteurized bacteria.
  • the extract of Akkermansia Mucinphila bacteria is Amuc_1100 protein.
  • the preparation method of Amuc_1100 protein is:
  • the resulting PCR product was cloned into the pET32aTEV vector by enzyme digestion method, and the expression plasmid of Amuc_1100 fused with His tag was constructed and transformed into E. coli C43 (DE3) Competent.
  • the present invention has the following advantages:
  • the present invention provides a new application of Akkermansia Mucinifila in antidepressant drugs through a large number of animal experiment screening and in-depth research.
  • the present invention uses bacterial knockout and fecal bacteria transplantation technology.
  • the experimental results show that Akkermansia Mucinifila transplantation can increase the movement distance of mice and reduce the immobility time of tail suspension experiments and forced swimming experiments, and significantly alleviate the depression state of mice.
  • Akkermansia Mucinifila has a good antidepressant effect and can be used clinically to prepare drugs for the treatment of depression. And experiments found that it has a good preventive effect on enteritis and depression model mice.
  • the present invention found that live Akkermansia Mucininiphila bacteria or pasteurized dead bacteria are effective and safe for patients with metabolic diseases. After comparison, the present invention is found to be equally safe for depressed patients, and live and dead bacteria are equally effective.
  • Figure 1 is a graph showing the influence of depression-like state in mice caused by chronic restraint stress on the relative abundance of the flora and Akkermansia Mucinifila.
  • Figure 2 is a graph of the results of the open field experiment of Example 1. (Compared with normal group, *P ⁇ 0.05; compared with depression group, # P ⁇ 0.05)
  • FIG. 4 is a view illustrating the results of forced swimming test (compared to normal group, * P ⁇ 0.05; compared with depression group, # P ⁇ 0.05).
  • Fig. 5 is a graph showing the results of the open field experiment of Example 2.
  • Figure 6 is a graph showing the results of the tail suspension experiment in Example 2.
  • Figure 7 is a graph showing the results of the forced swimming experiment in Example 2.
  • Fig. 8 is a graph showing the experimental results of the colon length of mice in each group in Example 2.
  • step 2 Place the AKK bacterial liquid obtained in step 1 in a water bath and heat to 62-65°C for 30 minutes.
  • the resulting PCR product was cloned into the pET32aTEV vector by enzyme digestion method, and the expression plasmid of Amuc_1100 fused with His tag was constructed and transformed into E. coli C43 (DE3) Competent.
  • Broad-spectrum antibiotics include Ampicillin (Ampicillin, 1g/l), Metronidazole (1g/l), Neomycin (Neomycin, 1g/l) or Vancomycin (Vancomycin, 500mg/l).
  • mice were randomly divided into 5 groups, each with 10 mice, namely the normal group, depression group, AKK treatment group, and pasteurized AKK bacteria treatment Group and Amuc_1100 protein treatment group, normal group mice were given 0.1ml of healthy human fecal bacteria and 0.1ml of sterile PBS solution daily; depression group mice were given 0.1ml of depressed patients' fecal bacteria and 0.1ml of sterile PBS solution daily; AKK The mice in the treatment group were given 0.1ml of fecal bacteria and 0.1ml of AKK bacteria solution for depression patients, and the mice in the pasteurized AKK group were given 0.1ml of fecal bacteria and 0.1ml pasteurized AKK bacteria solution, Amuc_1100 protein.
  • mice in the treatment group were given 0.1ml of fecal bacteria and Amuc_1100 protein (3 ⁇ g/mouse) to depression patients every day. Lasts for 14 days. After the fecal bacteria transplantation model was completed, the mice were subjected to behavioral testing. Behavioral testing included: open field experiment, tail suspension experiment and forced swimming experiment.
  • the tape was placed about 2 cm from the tail end, and the mouse was hung 50 cm from the ground. Each mouse was placed for 6 minutes, and the immobility time of the mouse was recorded in seconds in the last 4 minutes. Immobility time is defined as behavior without escape orientation. When the mice do not have any physical movement, they are considered static. .
  • mice were placed in a 30cm ⁇ 45cm water tank, and the water temperature was kept at 27°C. Each mouse was placed for 6 minutes, and the immobility time of the mouse was recorded in seconds in the last 4 minutes. Immobility time refers to the time during which an animal can float in the water without struggling and can keep its head above the water surface with only a few movements. Subsequently, the experimental mice were placed in a heating lamp to dry.
  • CRS chronic restraint stress depression
  • Extraction tool (Omega Bio-tek, Norcross, GA, US) to extract DNA from the stool samples of the above-mentioned clinically healthy people and depressed patients and the cecal contents of depressed mice replicated by the chronic restraint stress experiment.
  • a Nano-Drop 1000 spectrophotometer (Thermo Scientific Inc., Wilmington, DE, USA) was used to determine the DNA concentration.
  • the variable region of bacterial 16s rRNA gene V3-V4 was amplified by primers F338 (5'-ACTCCTACGGGAGGCAGCA-3') and R806 (5'-GGACTACHVGGGTWTCTAAT-3'). There are 3 replicates for each sample.
  • the polymerase chain reaction (PCR) products of the same sample are mixed and detected by 2% agarose gel electrophoresis.
  • the AxyPrep DNA Gel Recovery Kit (AXYGEN) is used to cut the gel and recover the PCR.
  • the product was eluted by Tris_HCl; detected by 2% agarose electrophoresis.
  • the amplicon was extracted from 2% agarose gel. All PCR products were purified by AxyPrepDNA gel extraction equipment (Axygen Biosciences). With reference to the preliminary quantitative results of electrophoresis, the PCR products were detected and quantified with QuantiFluor TM -ST blue fluorescence quantitative system (Promega), and then mixed in corresponding proportions according to the sequencing volume requirements of each sample.
  • the quality control filtering criteria are: (1) Filter the bases with a mass value of less than 20 at the tail of the read, and set a 50bp window. If the average quality value in the window is less than 20, the back-end bases will be truncated from the window and filtered after quality control.
  • the research of the present invention shows that Akkermansia Mucinphila is not only effective for patients with depression, but also effective for depression of the general population.
  • CRS chronic restraint stress
  • Broad-spectrum antibiotics include Ampicillin (Ampicillin, 1g/l), Metronidazole (1g/l), Neomycin (Neomycin, 1g/l) or Vancomycin (Vancomycin, 500mg/l).
  • mice are randomly divided into 3 groups, each with 10 mice, namely the normal group, depression group, AKK treatment group, and normal group mice
  • Normal mice were given 0.1ml of fecal bacteria and 0.1ml of sterile PBS solution daily; mice in the depression group were given 0.1ml of CRS mouse fecal bacteria and 0.1mL of sterile PBS solution in the above 1 daily; mice in the AKK treatment group were given daily CRS mice were given 0.1ml of fecal bacteria and 0.1ml of AKK bacterial solution.
  • the mice were subjected to behavioral testing. Behavioral testing included: open field experiment, tail suspension experiment and forced swimming experiment.
  • mice were given drinking water containing 2.5% DSS for 7 days to construct a mouse model of enteritis and depression. Then the mice were sacrificed, blood and colorectal tissue were taken, and the colon length was measured. Fix with 10% neutral formalin, conventionally sample, dehydrate, and embed in paraffin. The sections were treated with HE staining.
  • the safety test before and after transplantation of Akkermansia Mucinphila bacteria in mice indicates that the transplantation of the bacteria is safe and effective.
  • the present invention detects the blood routine, liver function, kidney function, spleen pathology, liver pathology of mice before and after transplantation of AkkermansiaMuciniphila bacteria, and no toxic side effects are found.
  • the research of the present invention based on the above method shows that Akkermansia Muciniphila has a good prevention and treatment effect on depression complicated with enteritis.

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Abstract

提供了一种Akkermansia Muciniphila菌在制备防治抑郁症的药物或保健品中的应用。所述Akkermansia Muciniphila菌在抑郁发病和抗抑郁中的作用，显示抑郁症患者肠道内的Akkermansia Muciniphila丰度明显降低。动物实验结果显示，抑郁小鼠肠道Akkermansia Muciniphila丰度显著降低。对抑郁小鼠模型给予Akkermansia Muciniphila菌移植，实验表明，Akkermansia Muciniphila菌移植可提升小鼠的运动距离并减少悬尾实验和强迫游泳实验的不动时间，显著缓解小鼠的抑郁状态，显示出了很好的抗抑郁疗效，且未见副作用。

Description

Akkermansia Muciniphila在制备抗抑郁药物或者保健品中的应用 技术领域
本发明涉及一种益生菌，具体涉及Akkermansia Muciniphila在抗抑郁药物或者保健品中的新用途，属于医药技术领域。
背景技术
抑郁症是以持久而显著的心境低落为主要临床特征的精神类疾病。其临床常伴有心境低落，自卑抑郁，甚至自杀企图或行为，并且多数患者有反复发作的倾向。因此，抑郁症是一种高致残率的疾病。根据世界卫生组织(WHO)报道，在一般人群中每100人就有15人受到过抑郁症的困扰。每年用于抑郁症治疗的费用占医疗总支出的12.3％。而在中国，估计有2000万-5000万的抑郁症患者，并且患病率逐年增长，目前市场尚未安全有效的抗抑郁生物制剂。
尽管抑郁症的发病与遗传、神经内分泌、神经免疫、体液等因素密切相关，但是其确切的病因并不清楚。近年来大量研究表明，肠道菌群在抑郁症发病过程中扮演着重要的角色。肠道菌群通过“肠-脑轴”影响着大脑的功能及行为。临床研究发现，抑郁症患者粪便中放线菌,乳杆菌,链球菌，尾蚴梭菌等细菌显著升高以及拟杆菌,酸球菌，韦荣球菌等细菌显著降低，动物实验研究表明，将抑郁患者的粪便移植给无菌小鼠，小鼠呈现抑郁样行为。
Akkermansiamuciniphila是一种2004年被鉴定出来的菌种，是人类消化道中普遍存在的共生菌，约占比3-5％。Akkermansiamuciniphila已被证实与肥胖,二型糖尿病,孤独症等慢性疾病密切相关。然而,还未见其在防治抑郁症中的报道。
发明内容
发明目的：本发明在Akkermansiamuciniphila现有用途的基础上，通过临床检测数据分析和动物实验筛选其临床功效，开发其在制备防治抑郁症及其抑郁症合并其它疾病的药物或保健品中的新应用。
技术方案：为了实现以上目的，本发明采取的技术方案为：
Akkermansia Muciniphila菌在制备预防和治疗抑郁症的药物或保健品中的应用。
Akkermansia Muciniphila菌在制备预防和治疗肠道疾病合并抑郁症的药物或保健品中的应用。作为优选，所述的肠道疾病包括肠炎、肠道肿瘤。
Akkermansia Muciniphila菌在制备预防和治疗糖尿病合并抑郁症的药物或保健品中的应用。
Akkermansia Muciniphila菌在制备预防和治疗高血压合并抑郁症的药物或保健品中的应用。
Akkermansia Muciniphila菌在制备预防和治疗肥胖合并抑郁症的药物或保健品中的应用。
Akkermansia Muciniphila菌在制备预防和治疗癫痫合并抑郁症的药物或保健品中的应用。
一种具有防治抑郁症的生物制剂，它包括Akkermansia Muciniphila菌或者Akkermansia Muciniphila菌提取物。
作为优选，以上所述的应用，所述的Akkermansia Muciniphila菌包括Akkermansia Muciniphila活菌或巴氏消毒后的菌。
一种具有防治抑郁症的生物制剂，所述的Akkermansia Muciniphila菌的提取物为Amuc_1100蛋白。
作为优选方案，Amuc_1100蛋白的制备方法为：
通过扩增编码1100胞内部分的基因，将所得的PCR产物通过酶切酶连的方法克隆到pET32aTEV载体上，构建融合His标签的Amuc_1100的表达质粒,并将其转化到大肠杆菌C43(DE3)感受态中.将该菌株接种在含有100ug/ml氨苄西林的LB培养液中，37℃、1800rpm培养，在指数中期(0D 600＝1.0)时添加0.5mMIPTG，28℃、180rpm诱导3h；4000rpm离心10分钟沉淀细胞，Bindingbuffer重悬沉淀细胞，并加入终浓度为1mM的PMSF，使用高压均质机将细胞裂解；将细胞裂解液离心12000rpm、30min、4℃，收集上清；通过Ni-16NTA His·Bind树脂亲和层析纯化得到Amuc_1100蛋白。
有益效果：本发明和现有技术相比具有以下优点：
(1)本发明在临床检测数据分析基础上,通过大量的动物实验筛选,深入研究，提供Akkermansia Muciniphila在抗抑郁药物中的新用途。本发明采用菌群敲除和粪菌移植技术,实验结果表明,Akkermansia Muciniphila移植可以提升小鼠的运动距离并减少悬尾实验和强迫游泳实验的不动时间，显著缓解小鼠的抑郁状态,提示Akkermansia Muciniphila有很好的抗抑郁疗效,临床上可以用于制备治疗抑郁症的药物。并且实验发现对肠炎合并抑郁模型小鼠具有很好的防治作用。
(2)实验结果表明Akkermansia Muciniphila，疗效可靠，使用安全，无毒副作用，可以用于制备防治抑郁的药品或保健食品。
(3)本发明发现，Akkermansia Muciniphila活菌或经过巴氏消毒死菌，对于代谢疾病患者均显示较好有效，而且安全。本发明经过对比发现，对于抑郁患者同样安全，而且活菌和死菌同样有效。
附图说明
图1为慢性束缚应激导致小鼠抑郁样状态对菌群及Akkermansia Muciniphila相对丰度的影响图。
图2为实施例1旷场实验结果图。(正常组比较，*P<0.05；与抑郁组比较， #P<0.05)
图3为实施例1悬尾实验结果图(与正常组比较，*P<0.05；与抑郁组比较， #P<0.05)。
图4为实施例1强迫游泳实验结果图(与正常组比较，*P<0.05；与抑郁组比较， #P<0.05)。
图5为实施例2旷场实验结果图。
图6为实施例2悬尾实验结果图。
图7为实施例2强迫游泳实验结果图。
图8为实施例2各组的小鼠结肠长度的实验结果图。
具体实施方式
根据下述实施例，可以更好地理解本发明。然而，本领域的技术人员容易理解，实施例所描述的具体的物料配比、工艺条件及其结果仅用于说明本发明，而不应当也不会限制权利要求书中所详细描述的本发明。
实施例1
1、Akkermansia Muciniphila菌液的制备方法：
取1ml的Akkermansia Muciniphila菌液,加入10ml的脑心浸出液肉汤中,于37℃厌氧环境下培育72h后,将菌液离心(6000rpm)20分钟,分离上清液,用无菌PBS溶液将菌液调配至OD600＝1，得到Akkermansia Muciniphila菌液(简称AKK菌液)。
2、巴氏消毒AKK菌(死菌)制备方法
将步骤1得到的AKK菌液放置于水浴箱中加热到62-65℃，保持30分钟。
3、Amuc_1100制备方法
通过扩增编码1100胞内部分的基因，将所得的PCR产物通过酶切酶连的方法克隆到pET32aTEV载体上，构建融合His标签的Amuc_1100的表达质粒,并将其转化到大肠杆菌C43(DE3)感受态中.将该菌株接种在含有100ug/ml氨苄西林的LB培养液中，37℃、1800rpm培养，在指数中期(0D 600＝1.0)时添加0.5mMIPTG，28℃、180rpm诱导3h；4000rpm离心10分钟沉淀细胞，Bindingbuffer重悬沉淀细胞，并加入终浓度为1mM的PMSF，使用高压均质机将细胞裂解；将细胞裂解液离心12000rpm、30min、4℃，收集上清；通过Ni-16NTA His·Bind树脂亲和层析纯化得到Amuc_1100蛋白。
2、临床粪便样本制备方法：
选取已收集的健康及抑郁志愿者粪便各5份，对于每组粪便样本进行混样处理，将混样处理的粪便溶于无菌PBS中,将菌液离心(1000rpm)10分钟,取出上清备用。
3、肠道菌群敲除造模
取6-8周C57BL/6雄性小鼠每日常规饮用广谱抗生素，持续4周。广谱抗生素包含氨苄西林(Ampicillin，1g/l)、甲硝唑(Metronidazole，1g/l)、新霉素(Neomycin，1g/l)或万古霉素(Vancomycin，500mg/l)。
4、粪菌移植
在肠道菌群敲除造模结束后,将C57BL/6小鼠30只,随机分为5组,每组10只，分别为正常组、抑郁组、AKK治疗组、巴氏消毒AKK菌治疗组和Amuc_1100蛋白治疗组，正常组小鼠每日给予健康人粪菌0.1ml和无菌PBS溶液0.1ml；抑郁组小鼠每日给予抑郁患者粪菌0.1ml和无菌PBS溶液0.1ml；AKK治疗组小鼠每日给予抑郁患者粪菌0.1ml和AKK菌液0.1ml，巴氏消毒AKK菌治疗组小鼠每日给予抑郁患者粪菌0.1ml和巴氏消毒AKK菌液0.1ml，Amuc_1100蛋白治疗组小鼠每日给予抑郁患者粪菌0.1ml和Amuc_1100蛋白(3μg/只)。持续14天。在粪菌移植造模结束后对小鼠进行行为学检测，行为学检测包括：旷场实验,悬尾实验和强迫游泳实验。
5、小鼠行为学检测
(1)旷场实验
将小鼠放入黑色木箱中(45cmx45cmx45cm)小室的角落，适应1分钟后，用视频计算机跟踪系统记录之后5分钟所有自发活动。以总运动距离作为运动活动的指标。
(2)悬尾实验
胶带放置在距尾端约2厘米处，将小鼠悬挂在距离地面50cm处。每只小鼠放置6min，并在最后4min以秒为单位记录小鼠不动时间。不动时间被定义为没有逃避导向的行为。当小鼠没有任何身体运动时，它们被认为是静止的。。
(3)强迫游泳实验
小鼠放置在30cm×45cm水箱中，水温保持在27℃。每只小鼠放置6min，并在最后4min以秒为单位记录小鼠不动时间。不动时间是指动物在水中漂浮而不挣扎，只需做一些动作就能保持头部高于水面的时间。随后，实验小鼠被置于取暖灯下晾干。
6、构建慢性束缚应激模型
构建慢性束缚应激抑郁小鼠模型(Chronic restraint stress,CRS)。CRS是当前国内外较为公认的抑郁模型，具体的造模方法为：取雄性C57BL/6小鼠，年龄在6-8周。适应性喂养1周后，每日将小鼠置放在0.5mm直径，50ML的离心管内3h，持续30天。在第30天记录行为测试结果。在CRS造模期间，其余各组限制小鼠喂食和给水。
7、Akkermansia Muciniphila菌群样本提取
按照制造商的说明，应用
Figure PCTCN2020079604-appb-000001
提取工具(Omega Bio-tek,Norcross,GA,U.S.)，将上述的临床健康人群和抑郁患者粪便样本及慢性束缚应激实验复制的抑郁状态小鼠盲肠内容物样本进行DNA提取。应用Nano-Drop 1000分光光度计(Thermo Scientific Inc.,Wilmington,DE,USA)确定DNA浓度。通过F338(5′-ACTCCTACGGGAGGCAGCA-3′)和R806(5′-GGACTACHVGGGTWTCTAAT-3′)引物对细菌16s rRNA基因V3-V4可变区域进行扩增。每个样本3个重复，将同一样本的聚合酶链反应(Polymerase Chain Reaction，PCR)产物混合后用2％琼脂糖凝胶电泳检测，使用AxyPrepDNA凝胶回收试剂盒(AXYGEN公司)切胶回收PCR产物，Tris_HCl洗脱；2％琼脂糖电泳检测。扩增子从2％琼脂糖胶中提取。所有的PCR产物通过AxyPrepDNA凝胶萃取设备(Axygen Biosciences公司)进行提纯。参照电泳初步定量结果，将PCR产物用QuantiFluor TM-ST蓝色荧光定量系统(Promega公司)进行检测定量，之后按照每个样本的测序量要求，进行相应比例的混合。在Illumina MiSeq平台上对萃取的PCR产物进行等分子、250bp双端测序分析。原始焦磷酸测序序列上传到NCBI数据中心的数据库SRA(Sequence Read Archive)。
8、Akkermansia Muciniphila菌群分析
对上述7提取得到的菌群样本进行菌群分析；采用高质量的序列合并重叠生成fastq文件。通过QIIME(version 1.9.1)软件将输出的fastq文件进行多路解码和质控过滤。质控过滤标准为：(1)过滤read尾部质量值20以下的碱基，设置50bp的窗口，如果窗口内的平均质量值低于20，从窗口开始截去后端碱基，过滤质控后50bp以下的read；(2)根据PE reads之间的overlap关系，将成对reads拼接(merge)成一条序列，最小overlap长度为10bp；(3)拼接序列的overlap区允许的最大错配比率为0.2，筛选不符合序列；(4)根据序列首尾两端的barcode和引物区分样品，并调整序列方向，barcode允许的错配数为0，最大引物错配数为2；应用UCHIME软件将嵌合体序列去除，并通过UPARSE(version 7.1http:// drive5.com/uparse/)软件将97％相似性的截点聚合生成OUTs。通过RDA分类器(http://rdp.cme.msu.edu/)并和silva(SSU115)16S rRNA数据库比对将每个OUT(门水平和属水平)进行比对分类。
9、统计分析
数据采用
Figure PCTCN2020079604-appb-000002
表示，采用单因素方差分析(ANOVA)进行检验，p<0.05为差异有统计学意义。
10、实验结果
(1)按以上菌群分析方法，检测分析健康人群和抑郁患者的Akkermansia Muciniphila相对丰度。如表1所示，抑郁患者Akkermansia Muciniphila较健康人群丰度均值显著降低，提示Akkermansia Muciniphila降低是患者抑郁致病的因素之一。
表1.抑郁症对菌群中Akkermansia Muciniphila相对丰度的影响
Figure PCTCN2020079604-appb-000003
*p<0.05与健康人群比较
(2)如表2和图1所示，慢性束缚应激实验复制小鼠抑郁状态，菌群检测发现Akkermansia Muciniphila菌丰度显著减低，进一步验证Akkermansia Muciniphila降低可能是患者抑郁致病主要因素之一。
表2慢性束缚应激导致小鼠抑郁样状态对菌群及Akkermansia Muciniphila相对丰度的影响
Figure PCTCN2020079604-appb-000004
*p<0.05与空白组比较
(3)抑郁患者粪菌移植实验发现，移植小鼠出现抑郁状态，提示肠道菌群与抑郁关系密切，但在口服上述AKK菌后，能有效改善小鼠的抑郁状态。
如图2所示，在旷场实验中，与正常组比较，抑郁组小鼠运动总距离显著降低(P<0.05)。与抑郁组比较，AKK菌治疗组、巴氏消毒AKK菌治疗组和Amuc_1100蛋白治疗组小鼠运动总距离显著增加(P<0.05)。
如图3所示，在悬尾实验中，与正常组比较，抑郁组小鼠的不动时间显著升高(P<0.05)，与抑郁组比较，AKK菌治疗组、巴氏消毒AKK菌治疗组和Amuc_1100蛋白治疗组小鼠不动时间显著降低(P<0.05)。
如图4所示，在强迫游泳实验中，与正常组比较，抑郁组小鼠的不动时间显著升高(P<0.05)，与抑郁组比较，AKK菌治疗组、巴氏消毒AKK菌治疗组和Amuc_1100蛋白治疗组小鼠不动时间显著降低(P<0.05)。
如上述实验结果表明，将AKK菌缺乏的抑郁患者粪便移植给肠道菌群敲除鼠，小鼠 呈抑郁样状态。经口服AKK活菌和巴氏消毒AKK菌及其Amuc_1100蛋白治疗后，小鼠的抑郁状态显著改善。以上结果表明，本发明提供的AKK菌及其Amuc_1100蛋白具有很好的抗抑郁的功效。本发明筛选的Akkermansia Muciniphila菌或巴氏消毒后的死菌不仅可以作为食品、保健品中使用，也可以用于制备药品，既有治疗作用，也有预防作用。
本发明研究表明，Akkermansia Muciniphila菌不仅对抑郁症患者有效，而且对一般人群抑郁状态有效。
实施例2考察AKK菌对抑郁合并肠炎模型小鼠的治疗作用
1、构建慢性束缚应激抑郁小鼠模型(Chronic restraint stress,CRS)CRS具体的造模方法为：取雄性C57BL/6小鼠，年龄在6-8周。适应性喂养1周后，每日将小鼠置放在0.5mm直径，50ML的离心管内3h，持续30天。在第30天记录行为测试结果。在CRS造模期间，其余各组限制小鼠喂食和给水；实验结束对所有小鼠进行行为学检测，行为学检测包括：旷场实验,悬尾实验和强迫游泳实验。行为学检测显示抑郁造模成功后，立即处死小鼠并取出盲肠内容物。随机将3只小鼠的盲肠内容物进行混样处理，溶于无菌PBS中,将菌液离心(1000rpm)10分钟,取出上清备用。
2、肠道菌群敲除造模
取6-8周C57BL/6雄性小鼠每日常规饮用广谱抗生素，持续4周。广谱抗生素包含氨苄西林(Ampicillin，1g/l)、甲硝唑(Metronidazole，1g/l)、新霉素(Neomycin，1g/l)或万古霉素(Vancomycin，500mg/l)。
3、粪菌移植(FMT)
3.1、在肠道菌群敲除造模结束后，将C57BL/6小鼠30只，随机分为3组，每组10只，分别为正常组、抑郁组、AKK治疗组，正常组小鼠每日给予正常小鼠粪菌0.1ml和无菌PBS溶液0.1ml；抑郁组小鼠每日给予上述1的CRS小鼠粪菌0.1ml和无菌PBS溶液0.1mL；AKK治疗组小鼠每日给予CRS小鼠粪菌0.1ml和AKK菌液0.1ml。持续14天。在粪菌移植造模结束后对小鼠进行行为学检测，行为学检测包括：旷场实验、悬尾实验和强迫游泳实验。
3.2、行为学检测实验结束后，将上述30只C57BL/6小鼠，给与含2.5％DSS的饮用水自由饮水7天，构建肠炎合并抑郁小鼠模型。然后处死小鼠，取血、结直肠组织，测量结肠长度。用10％中性福尔马林固定，常规取材，脱水，石蜡包埋。切片经HE染色等处理。
4、实验结果
4.1行为学检测结果：
如图5旷场实验，经粪菌移植(FMT)，和正常组比较，抑郁组小鼠运动总距离显著降低。与抑郁组比较，AKK组小鼠运动总距离显著增加。(P＜0.05)。
如图6悬尾实验，经粪菌移植(FMT)，和正常组比较，抑郁组小鼠不动时间显著增加。和抑郁组比较，AKK组小鼠不动时间显著降低。(P＜0.05)。
如图7强迫游泳实验，经粪菌移植(FMT)，和正常组比较，抑郁组小鼠不动时间显著增加。和抑郁组比较，AKK组小鼠不动时间显著降低。(P＜0.05)
4.2对肠炎检测结果
如图8所示，经粪菌移植(FMT)和DSS诱导肠炎，和正常小鼠造模的肠炎组比较，抑郁组肠炎小鼠的结肠长度显著降低。和抑郁组肠炎小鼠比较，AKK治疗组小鼠结肠长度显著增加(P＜0.05)。
且由结肠病理观察结果表明，单个肠炎组小鼠结肠炎症严重程度为轻至中度；而抑郁组肠炎小鼠结肠炎症严重程度为中度，多数累及粘膜层和粘膜下层，个别的累及肌层，炎症严重程度为中度，炎细胞类型主要为单核巨噬细胞和中性粒细胞及嗜酸性粒细胞。而AKK治疗组结肠炎症较抑郁组明显减轻，粘膜下层、肌层、浆膜层无明显充血、水肿，无明显炎细胞浸润。
本发明实验过程中，小鼠移植AkkermansiaMuciniphila菌前后安全性检测提示该菌种移植安全有效。本发明对AkkermansiaMuciniphila菌移植前后小鼠血常规、肝功能、肾功能、脾脏病理、肝脏病理进行了检测，未发现毒副作用。
本发明依据上述方法研究表明，Akkermansia Muciniphila菌的对抑郁症合并肠炎具有很好的防治效果。
以上实施方式只为说明本发明的技术构思及特点，其目的在于让熟悉此项技术的人了解本发明内容并加以实施，并不能以此限制本发明的保护范围，凡根据本发明精神实质所做的等效变化或修饰，都应涵盖在本发明的保护范围内。

Claims (12)

  1. Akkermansia Muciniphila菌在制备预防和治疗抑郁症的药物或保健品中的应用。
  2. Akkermansia Muciniphila菌在制备预防和治疗肠道疾病合并抑郁症的药物或保健品中的应用。
  3. Akkermansia Muciniphila菌在制备预防和治疗糖尿病合并抑郁症的药物或保健品中的应用。
  4. Akkermansia Muciniphila菌在制备预防和治疗高血压合并抑郁症的药物或保健品中的应用。
  5. Akkermansia Muciniphila菌在制备预防和治疗肥胖合并抑郁症的药物或保健品中的应用。
  6. Akkermansia Muciniphila菌在制备预防和治疗癫痫合并抑郁症的药物或保健品中的应用。
  7. Akkermansia Muciniphila菌在制备预防和治疗产后抑郁症的药物或保健品中的应用。
  8. 根据权利要求2所述的Akkermansia Muciniphila菌在制备预防和治疗肠道疾病合并抑郁症的药物或保健品中的应用，其特征在于，所述的肠道疾病包括肠炎、肠道肿瘤。
  9. 根据权利要求1至8任一项所述的应用，其特征在于，所述的Akkermansia Muciniphila菌包括Akkermansia Muciniphila活菌或巴氏消毒后的菌。
  10. 一种具有防治抑郁症的生物制剂，其特征在于，它包括Akkermansia Muciniphila菌或基于Akkermansia Muciniphila菌的提取物。
  11. 根据权利要求10所述的具有防治抑郁症的生物制剂，其特征在于，所述的Akkermansia Muciniphila菌的提取物为Amuc_1100蛋白。
  12. 根据权利要求11所述的具有防治抑郁症的生物制剂，其特征在于，Amuc_1100蛋白的制备方法为:
    通过扩增编码1100胞内部分的基因，将所得的PCR产物通过酶切酶连的方法克隆到pET32aTEV载体上，构建融合His标签的Amuc_1100的表达质粒,并将其转化到大肠杆菌C43(DE3)感受态中.将该菌株接种在含有100ug/ml氨苄西林的LB培养液中，37℃、1800rpm培养，在指数中期(0D 600＝1.0)时添加0.5mM IPTG，28℃、180rpm诱导3h；4000rpm离心10分钟沉淀细胞，Bindingbuffer重悬沉淀细胞，并加入终浓度为1mM的PMSF，使用高压均质机将细胞裂解；将细胞裂解液离心12000rpm、30min、4℃，收集上清；通过Ni-16NTA His·Bind树脂亲和层析纯化得到Amuc_1100蛋白。
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CN111690044A (zh) * 2020-05-06 2020-09-22 中南大学湘雅二医院 Amuc1100蛋白的用途
CN115252749A (zh) * 2022-03-26 2022-11-01 吉林大学 附子理中制剂作为抗抑郁靶向药物的应用
BE1029502B1 (fr) * 2021-08-19 2023-01-17 The Akkermansia Company Composition comprenant de l'Akkermansia muciniphila pasteurisée pour le traitement ou la prévention de l'anxiété liée au syndrome du côlon irritable
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BE1029502B1 (fr) * 2021-08-19 2023-01-17 The Akkermansia Company Composition comprenant de l'Akkermansia muciniphila pasteurisée pour le traitement ou la prévention de l'anxiété liée au syndrome du côlon irritable
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CN115252749A (zh) * 2022-03-26 2022-11-01 吉林大学 附子理中制剂作为抗抑郁靶向药物的应用
CN117065002A (zh) * 2023-02-02 2023-11-17 浙江大学 阿克曼菌膜蛋白Amuc_1100在预防和/或治疗年龄相关性认知下降产品制备中的应用
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