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WO2013015331A1 - Animal modèle de stéato-hépatite non alcoolique (nash) - Google Patents

Animal modèle de stéato-hépatite non alcoolique (nash) Download PDF

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WO2013015331A1
WO2013015331A1 PCT/JP2012/068878 JP2012068878W WO2013015331A1 WO 2013015331 A1 WO2013015331 A1 WO 2013015331A1 JP 2012068878 W JP2012068878 W JP 2012068878W WO 2013015331 A1 WO2013015331 A1 WO 2013015331A1
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model animal
nash
nash model
animal according
fat diet
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PCT/JP2012/068878
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Japanese (ja)
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憲広 久保田
範江 増岡
狩野　光芳
将一 角
稔秀 小林
百合子 永田
幸司 宮崎
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株式会社ヤクルト本社
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/20—Animals treated with compounds which are neither proteins nor nucleic acids
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/25—Animals on a special diet
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0362—Animal model for lipid/glucose metabolism, e.g. obesity, type-2 diabetes

Definitions

  • the present invention relates to a NASH model animal showing a pathological condition similar to human non-alcoholic steatohepatitis (NASH) and a method for producing the same.
  • NASH non-alcoholic steatohepatitis
  • NASH nonalcoholic fatty liver disease
  • Non-patent Document 1 methionine and / or choline-deficient diet models
  • nitrite and hydroxylamine are administered to develop methemoglobinemia, and hypoxia in vivo
  • the model (patent document 2) etc. which form is known these cannot be said to reflect the pathological condition of NASH histologically.
  • Patent Document 3 a model mouse to which an N-acetyl- ⁇ -D-glucosaminidase inhibitor such as streptozotocin is administered
  • Patent Document 4 a model mouse to which a high fat diet and a tetracycline antibiotic are administered
  • Patent Document 2 a model mouse in which carbon tetrachloride is administered once.
  • the pathological findings of these animal models show only large fatty liver, and there is no inflammation around the central lobule vein and balloon-like swelling of hepatocytes, which is characteristic of NASH.
  • This animal model is hardly a human NASH model. Therefore, the subject of this invention is providing the NASH model animal which produces the pathological condition peculiar to human NASH, and its production method.
  • a drug that induces hepatic disorder while ingesting a high-fat diet to obesity (hereinafter referred to as a “health disorder”) ,
  • a hepatic disorder drug) is a large drop of fatty liver, inflammation around the central vein in the leaflet of the liver, and hepatocytes are swollen like a balloon.
  • the present inventors have found that a model animal having a specific pathological condition can be prepared, and that the use of the model animal can evaluate the NASH preventive and therapeutic effect of the test substance and can screen for NASH preventive and therapeutic agents.
  • the present invention provides a NASH model animal obtained by ingesting a high fat diet and administering a hepatotoxic agent four times or more.
  • the present invention also provides a method for producing a NASH model animal, characterized by ingesting a high fat diet and administering a hepatic disorder agent four or more times.
  • the present invention also provides a screening method for a NASH prophylactic / therapeutic agent characterized by using the NASH model animal.
  • the present invention provides a method for evaluating the NASH preventive and therapeutic effect of a test substance, characterized by using the NASH model animal.
  • the present invention also provides a NASH model animal which is a large fatty liver, has inflammation around the central vein in the lobule of the liver, and hepatocytes are swollen like a balloon.
  • the NASH model animal of the present invention is a large-scale fatty liver, has inflammation around the central vein in the lobule of the liver, and has the symptoms peculiar to human NASH that hepatocytes are swollen like a balloon. It is useful as a human NASH model. By using the NASH model animal of the present invention, it becomes possible to screen for drugs and foods effective for human NASH. It can also be used to evaluate NASH preventive and therapeutic effects of test substances.
  • A Group 1 (untreated control group).
  • B Group 2 (high fat diet control group).
  • C Group 3 (high fat diet + carbon tetrachloride once administration group).
  • DF Group 9 (high fat diet + carbon tetrachloride 8 administration group).
  • A Histological image of normal liver.
  • B Small fat droplets in hepatocytes observed from around the central vein to the middle leaflet zone.
  • C Small fat droplets observed from around the central vein to the middle leaflet zone as in the high fat diet control group.
  • D Large drop fatty liver (arrowhead).
  • E Balloon-like swelling of the hepatocytes observed around the central vein (arrow).
  • F Lymphocyte-dominated inflammatory cell infiltration around the central vein. *: Central vein. #: Portal area.
  • B Group 2 (high fat diet control group).
  • C Group 3 (high fat diet + carbon tetrachloride once administration group).
  • D Group 9 (high fat diet + carbon tetrachloride 8 dose group).
  • FIGS. 8A to 8C are diagrams showing fibrosis caused by administration of the high fat diet and carbon tetrachloride (0.25 mL / kg body weight) 8 times in Example 2.
  • FIG. A Large droplet fatty liver; Large droplet fat accumulates in hepatocytes from the central part of the leaflet to the peripheral part.
  • B Cytoplasmic vacuoles are positive for Sudan III staining (fat).
  • C Minor fibrosis around the central vein. *: Central vein.
  • A x10.
  • B, C x20.
  • A HE staining.
  • B Sudan III staining (frozen section).
  • C MT staining.
  • AB is a figure which shows the inflammatory condition by the high fat diet of Example 2, and carbon tetrachloride (0.25 mL / kg body weight) 8 times administration.
  • A Infiltration of inflammatory cells around the central vein.
  • B Balloon-like swelling of the hepatocytes observed around the central vein (arrow). *: Central vein.
  • B x40.
  • A, B HE staining. It is a figure which shows the histopathological change in the liver of a microbial cell test group.
  • A, B Normal diet group (normal liver).
  • C, D Control group (NASH).
  • E, F L. plantarum group (findings in which inflammation in leaflets was slightly suppressed).
  • G, H L.G. salivarius group (findings in which inflammation in leaflets was slightly suppressed).
  • AH x20.
  • B, D, F, H MT staining.
  • the NASH model animal of the present invention can be prepared by ingesting a high fat diet and administering a hepatopathy agent four times or more.
  • mice are not particularly limited, and examples include mice, rats, guinea pigs, hamsters, rabbits, monkeys, etc., but it is preferable to use mice.
  • a mouse it may be a normal mouse and may be a hybrid or strain, but a mouse used as an experimental animal, for example, C57BL / 6 mouse, C3H mouse, ICR mouse, BALB / c line A mouse or the like is preferable.
  • the high-fat diet may be a diet containing 20% by mass or more of fat, and a diet containing 30 to 80% by mass of fat, particularly a diet containing 40 to 60% by mass of fat is preferable.
  • fat all kinds of fat can be used regardless of animal fat and vegetable fat, and examples thereof include corn oil, soybean oil, rapeseed oil, beef tallow, and lard. Pork fat is preferred.
  • components other than fat in the high-fat diet may be normal dietary components, and may include proteins, carbohydrates, minerals, and the like.
  • the intake period of the high fat diet is not particularly limited as long as it is necessary for obesity to occur, but is preferably 2 weeks or more, more preferably 4 weeks or more, further 4 to 16 weeks, and particularly preferably 8 to 16 weeks. .
  • the start time of high fat diet intake is not particularly limited, but is preferably from 5 weeks of age. Moreover, what is necessary is just to let an animal ingest a high fat diet freely every day.
  • the hepatic disorder used is not particularly limited, and examples thereof include carbon tetrachloride, galactosamine, acetaminophen, LPS (lipopolysaccharide), and carbon tetrachloride is particularly preferable.
  • the administration frequency is preferably 4 to 10 times, more preferably 4 to 8 times. When the number of administrations is less than 4, no inflammation or fibrosis occurs in the liver, and symptoms unique to human NASH do not develop.
  • the means for administering the hepatic disorder agent is not particularly limited, such as intraperitoneal administration, intravenous administration, oral administration, and subcutaneous administration, but subcutaneous administration is preferred.
  • 0.05 to 0.5 mL / kg body weight / time is preferable, and 0.1 to 0.25 mL / kg body weight / time is more preferable. If it is less than 0.05 mL / kg body weight / time, it may not exhibit NASH symptoms, and if it is more than 0.5 mL / kg body weight / time, it does not develop symptoms peculiar to human NASH and normal liver damage.
  • the start time of administration of the hepatic disorder is not particularly limited, but is preferably 2 weeks after the start of high fat diet intake, more preferably 4 weeks after the start of high fat diet, and more preferably 8 weeks. Later.
  • the administration interval of the hepatotoxic agent is preferably 1 to 3 times a week, particularly preferably twice a week. In addition, it is preferable to continue ingesting a high fat diet during the period of administration of the hepatopathy agent.
  • More preferable high fat diet intake and hepatotoxic agent administration schedule is to take a high fat diet for 8 weeks or more and then continue to consume the high fat diet twice a week with 0.1 to 0 hepatotoxic agent 25 mL / kg body weight / dose, 4 times or more in total.
  • a high-fat diet is ingested and a hepatotoxic agent is administered four or more times, pathological findings peculiar to human NASH appear in animals.
  • the pathological condition is macroscopic fatty liver, inflammation around the central vein in the leaflet of the liver, and hepatocytes swollen like balloons.
  • the NASH model animal of the present invention is particularly useful as a pathological model of human NASH.
  • the balloon-like swelling of the hepatocytes is not particularly limited in the place where the balloon-like swelling occurs, but it may occur around the central vein because it is more similar to the symptoms of human NASH.
  • Fatty liver means a state in which a large amount of fat is deposited in the liver, and more specifically, a state in which large fatty liver (deposition) and / or small fat accumulation (deposition) is observed.
  • large droplet fatty liver (deposition) needs to be observed in that it is more similar to human NASH.
  • the NASH model animal of the present invention preferably also exhibits a symptom that the liver tissue becomes fibrotic, and the NASH model animal that has developed until fibrosis is known as Matteoni et al. It can be a model animal of NASH that is well matched with NASH (Type 4) of the classification of the above, and has a more advanced disease state. In addition, it is more preferable that fibrosis occurs around the central vein because it is similar to the symptoms of human NASH.
  • the NASH model animal of the present invention is selected from serum aspartate aminotransferase (AST (GOT)), alanine aminotransferase (ALT (GPT)), free fatty acid (NEFA), and neutral fat (TG) 1
  • AST serum aspartate aminotransferase
  • ALT alanine aminotransferase
  • NEFA free fatty acid
  • TG neutral fat
  • the AST is 200 U / L or more, more preferably 500 U / L or more, and still more preferably 1000 U / L or more.
  • ALT is 300 U / L or more, more preferably 800 U / L or more, and still more preferably 1200 U / L or more.
  • Free fatty acid (NEFA) is 1000 ⁇ Eq / L or more, more preferably 1200 ⁇ Eq / L or more.
  • the neutral fat is 20 mg / dL or more, more preferably 30 mg / dL or more.
  • the NASH model animal of the present invention shows a pathological condition similar to the symptoms of human NASH.
  • the NASH model animal of the present invention exhibits symptoms specific to human NASH, and is useful for studying the pathology of NASH, as well as screening for NASH prophylactic and therapeutic agents (NASH prophylactic and therapeutic foods). Useful for. It is also useful for evaluating NASH preventive and therapeutic effects of test substances.
  • the NASH preventive and therapeutic effect of the test substance can be evaluated, and a NASH preventive and therapeutic agent can be screened.
  • the degree of liver damage can be determined by comparison with a test substance non-administered group and / or with a normal animal group (a normal diet group, a hepatotoxic agent non-administered group, etc.).
  • lactic acid bacteria are mentioned as an active ingredient of the NASH preventive and therapeutic agent screened by the screening method using the NASH model animal of the present invention.
  • a lactic acid bacterium has a lactic acid bacteria-containing high fat diet containing 1 ⁇ 10 10 / g of lactic acid bacteria for 12 weeks so that the daily intake is 3 g, and administration of the lactic acid bacteria-containing high fat diet is started.
  • Lactic acid bacteria when carbon tetrachloride (initial: 0.05 ml / kg body weight, second and subsequent times: 0.1 ml / kg body weight) was subcutaneously administered twice a week for a total of 8 times from week 9 to week 12 It is a lactic acid bacterium in which the value of serum ALT is reduced by 65% or more compared to non-administered mice.
  • thermophilus 2.6 g / day
  • tetrachloride from the 9th to 12th week from the start of administration of the high-fat diet containing bacterial powder Carbon (initial: 0.05 ml / kg body weight, second and later: 0.1 ml / kg body weight) is subcutaneously administered twice a week for a total of 8 times.
  • Blood is collected 24 hours after the final administration of carbon tetrachloride, and the ALT level in the serum is measured using transaminase CII test Wako (Wako Pure Chemical Industries).
  • lactic acid bacterium whose ALT value is reduced by 65% or more compared to a mouse not administered with lactic acid bacteria (a mouse administered with the high fat diet shown in Table 9 for 12 weeks and administered with carbon tetrachloride eight times in the same manner as described above). .
  • the average intake of feed per mouse in this lactic acid bacteria non-administered group is 2.5 g / day.
  • the intake per day is 3 g for 12 weeks to mice with a high fat diet containing 1 ⁇ 10 10 / g lactic acid bacteria.
  • Carbon tetrachloride (first time: 0.05 ml / kg BW, second time and later: 0.1 ml / kg BW) twice a week from the 9th to 12th week from the start of administration of the high-fat diet containing lactic acid bacteria, Even when administered subcutaneously a total of 8 times, the value of serum ALT is reduced by 65% or more compared to mice not administered with lactic acid bacteria. Moreover, it is more preferable to use a lactic acid bacterium in which the AST value in serum is reduced by 40% or more compared to a mouse not administered with a lactic acid bacterium. Preferably, the serum ALT value is decreased by 70% or more, more preferably 80% or more.
  • serum AST is reduced by 50% or more, more preferably 80% or more.
  • inflammation and fibrosis in the lobule which are symptoms unique to NASH, are preferably improved, and it has an excellent NASH preventive and therapeutic action.
  • These lactic acid bacteria can also be used as anti-inflammatory agents and anti-fibrotic agents in the leaflets.
  • Lactobacillus acidophilus Lactobacillus acidophilus
  • Lactobacillus helveticus Lactobacillus helveticus
  • Lactobacillus salivarus Lactobacillus salivarius
  • Bacillus gasseri Lactobacillus fermentum
  • Lactobacillus reuteri Lactobacillus reuteri
  • Lactobacillus delbruxy subspecies Bulgaricus (Lactobacillus delbrueckii subsp. Bulgaricus), Lactobacillus del bruecki subspecies.
  • Lactobacillus pulsium Lactobacillus pulsium, Lactobacillus pulsium, Lactobacillus johnsoniii, Lactobacillus johnsoniii, Lactobacillus johnsoniii Streptococcus bacteria such as Streptococcus salivarius), Lactococcus lactis subspecies. Lactococcus lactis subsp. Lactis, Lactococcus lactis subspecies. Lactococcus bacteria such as Lactococcus lactis subsp.
  • Cremoris Lactococcus raffinolactis, bacteria such as Enterococcus faecalis, Enterococcus ecterium c. ⁇ Breidobacterium breve, Bifidobacterium longum (Bifidobacterium longum), Bifidobacterium infantitis, Bifidobacterium address sentis (B) fibacterium adolescententis, Bifidobacterium bifidum, Bifidobacterium catenatumum, Bifidobacterumumumumum ), Bifidobacterium gallicum having a source isolated from the human intestine, Bifidobacterium lactis used in foods, Bifidobacterium galtis Idobakuteriumu animalis (Bifidobacterium animalis) lactic acid bacteria and the like.
  • Bifidobacterium bifidum Lactobacillus plantarum, Lactobacillus salivarius, Streptococcus thermophilus
  • Bifidobacterium bifidum YIT 10347 (FERM BP-10613: Date of deposit June 23, 2005), Lactobacillus plantarum YIT 0132 (FERM BP-11349: Date of deposit February 24, 2010), Lactobacillus salivaius YIT 0432 (FERM P-22129: date of deposit June 15, 2011), Streptococcus thermophilus YIT2001 (FERM BP-7538: date of deposit January 31, 2001 [2001])
  • At least one member selected from the group consisting of) is preferred.
  • the usage form of lactic acid bacteria can mention 1 or more chosen from the bacterial cell (live cell) and the processed material of the said bacterial cell, for example.
  • the treated product is not particularly limited as long as it is a treated product by a conventional method.
  • the treated cells dead cells
  • lyophilized products thereof cultures containing them
  • bacterial ultrasonic waves etc.
  • Crushing liquid, bacterial enzyme treatment liquid, solid residue separated by solid-liquid separation means such as filtration or centrifugation; treatment liquid from which cell wall has been removed by enzyme or mechanical means, concentrate of the treatment liquid, Diluted product, dried product thereof, etc .; Nucleic acid-containing fraction obtained by dissolving bacteria with a surfactant and then precipitating with ethanol, etc .;
  • separation / purification treatment such as separation by various chromatography.
  • the dead cells can be obtained, for example, by heat treatment, treatment with drugs such as antibiotics, treatment with chemical substances such as formalin, treatment with ultraviolet rays, treatment with radiation such as ⁇ rays.
  • the effectiveness can be expected regardless of whether it is a viable cell, dead cell, or other treated cell product, as long as it contains a certain number of viable cells at the end of cultivation of lactic acid bacteria.
  • the lactic acid bacteria for NASH preventive treatment can be provided.
  • use of the said lactic acid bacteria for NASH prophylactic / therapeutic agent manufacture can be provided.
  • use of the said lactic acid bacteria for NASH prevention treatment can be provided.
  • a NASH prophylactic treatment method characterized by ingesting an effective amount of the lactic acid bacteria can be provided.
  • the NASH preventive and therapeutic agent of the present invention can be used not only as a pharmaceutical product but also as a food / beverage product, a quasi-drug, a pet food and the like.
  • the number of lactic acid bacteria should be 1 ⁇ 10 9 or more.
  • the amount contained as a daily amount is preferable, the amount containing 5 ⁇ 10 9 to 2 ⁇ 10 12 as a daily amount is more preferable, and the amount containing 1 ⁇ 10 10 to 1 ⁇ 10 12 as a daily amount is further preferable.
  • the number of bacteria in the present invention is calculated based on the number of living bacteria at the end of lactic acid bacteria culture.
  • Example 1 Production of model animals The produced NASH model mice are shown below (Table 1). Carbon tetrachloride (0.1 mL / kg body weight) was subcutaneously administered to male C57BL / 6N mice fed with a high-fat diet (40% beef tallow mixed feed) and bred for 8 weeks under the following conditions. After the last administration, the blood was sacrificed under ether anesthesia. In addition, livers were collected from 5 animals in each group and fixed with 10% neutral buffered formalin. Table 2 shows the composition of the high fat diet. For Group 1 and Group 9, blood was collected from the abdominal aorta just prior to sacrifice. The obtained blood was left on ice for 1 hour or more, and then centrifuged at 3000 rpm for 15 minutes to prepare serum. When carbon tetrachloride was administered twice or more, the maximum number of administrations per week was set to twice.
  • Table 1 Carbon tetrachloride (0.1 mL / kg body weight) was subcutaneously administered to male C57BL / 6N mice
  • NASH requires large droplet fatty liver, lobular inflammation and balloon-like swelling of hepatocytes (Type 3 in Table 3), and in addition to these symptoms, when Mallory bodies or fibrosis is observed It is classified as Type 4.
  • hepatocytes Type 3 in Table 3
  • Mallory bodies or fibrosis is observed It is classified as Type 4.
  • inflammatory cell infiltration mainly consisting of lymphocytes and mild fibrosis were observed around the central vein ( FIG. 1F, FIG. 2D).
  • the incidence of inflammatory cell infiltration (inflammation) tended to increase in proportion to the number of administrations (Tables 4 and 5).
  • Fibrosis was observed around the central vein and surrounding hepatocytes (FIG. 2D), and was observed in each group at a rate of 3 to 4 out of 5 cases (Tables 4 and 5).
  • fibrosis images that progressed to bridging fibrosis as reported in human cirrhosis were not observed in all groups.
  • high fat diet + carbon tetrachloride 4 times administration group Group 7
  • high fat diet + carbon tetrachloride 6 times administration group Group 8
  • NASH diagnostic criteria Type 4 in Table 3 classified by Matteoni et al. It became clear.
  • NASH-like lesions are not induced in mice only by administration of a high-fat diet alone, but NASH-like lesions are also induced in the case of feeding a high-fat diet and single and double administration of carbon tetrachloride. It became clear that not. In addition, even after 72 hours from the single administration of carbon tetrachloride, the symptoms did not progress, and it became clear that NASH-like lesions were not induced. On the other hand, it became clear that NASH-like lesions were induced by administration of carbon tetrachloride four or more times, and that NASH-like lesions were induced strongly and frequently by increasing the number of administrations of carbon tetrachloride. .
  • NASH-like lesions can be induced by administering a hepatopathy agent at a dose lower than 1 ml / kg body weight / time, which is usually an amount that causes hepatopathy, and four times or more. It became clear. In view of the unclear generation mechanism of NASH, the reason why NASH-like lesions are induced by the above method is unclear.
  • Example 2 Except for the carbon tetrachloride dosage of 0.25 mL / kg body weight, the carbon tetrachloride was subcutaneously administered 8 times (2 times / week) in the same manner as in Example 1.
  • NASH model mice having the histopathological findings shown in Table 7 and the liver symptoms shown in FIGS. This mouse exhibited inflammation around the central vein, resulting in fibrosis, and large droplet fatty liver and hepatocyte balloon-like swelling.
  • Example 3 Test group and test schedule The following experiment was performed using C57BL / 6N strain, 7-week-old mice.
  • the normal diet group is a test group in which AIN93G, which is a general breeding feed, was ingested for 12 weeks.
  • AIN93G which is a general breeding feed
  • In the control group for the onset of NASH, intake of a high fat diet for 12 weeks and carbon tetrachloride (initial: 0.05 ml / kg body weight, second and subsequent: 0.1 ml / kg body weight) from week 9 to week 12 was administered subcutaneously a total of 8 times.
  • Each lactic acid bacteria administration group was ingested with a diet containing 0.05% of each freeze-dried bacterial powder based on the high fat diet of the control group (the number of bacteria in the feed: The number of bacteria in 1 g of feed is determined by the following formula: number of viable bacteria at the end of culture in culture medium / amount of bacterial powder produced by lyophilization (g) ⁇ weight of bacterial powder in 1 g of feed ( g) L.
  • Feed and drinking water were freely consumed in each test (average feed weight per mouse normal diet: 2.4 g / day, control: 2.5 g / day, L. Crispatus: 2.4 g / day)
  • Necropsy was performed 24 hours after the final administration of carbon tetrachloride, and blood and organs were collected. Serum was prepared from blood and used to measure AST and ALT. The collected liver was immersed and stored in a 10% neutral buffered formalin solution for histopathological analysis.
  • livers of each test group were used for histopathological analysis.
  • the liver fixed with a formalin solution was cut out by a conventional method, embedded in paraffin, and then the paraffin block was sliced into a thickness of about 4 ⁇ m, stained with Hematoxylin-Eosin (HE), and observed with an optical microscope.
  • HE Hematoxylin-Eosin
  • MT Masson's trichrome staining was performed on paraffin sections to prove collagen fibers. Intralobular inflammation and fibrosis were examined as diagnostic items for histopathology.
  • the grading of the pathological condition is that the entire visual field of the liver section is observed and no pathological condition is observed:-(negative), the pathological condition is 20% or less of the total visual field: ⁇ (very mild), the pathological condition is 20 to 50% of the total visual field : + (Mild), 50-80% of all visual fields: ++ (moderate), 80% or more of all visual fields: ++ (severe).
  • L. In the crisptus group neither AST nor ALT significantly decreased.
  • Table 10 shows the AST results.
  • Table 11 shows the results of ALT.
  • the number of bacteria that a mouse ingests per day is Bifidum: 2.12 ⁇ 10 9 , L. Plantarum: 3.2 ⁇ 10 9 , L. Salivarius: 9.62 ⁇ 10 8 , S. thermophilus: 2.45 ⁇ 10 8 . Since the weight of the mouse used was about 37 g, Bifidum: 5.73 ⁇ 10 10 / kg BW / day, L. Plantarum: 8.65 ⁇ 10 10 / kg BW / day, L.P. Sarivarius: 2.6 ⁇ 10 10 / kg BW / day, S. var. thermophilus: 6.62 ⁇ 10 9 / kg BW / day.
  • the lactic acid bacterium of the present invention has the advantage of exhibiting a higher effect with a smaller number of bacteria than the lactic acid bacterium described in JP-T-2005-512590.
  • NASH model animals can be used to screen for NASH preventive and therapeutic agents, and it has been shown that NASH preventive and therapeutic effects of test substances can also be evaluated.

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Abstract

L'invention concerne un animal modèle de NASH dans lequel les caractéristiques de pathologie de NASH chez des êtres humains sont produites, et un procédé de production de l'animal modèle de NASH. L'animal modèle de NASH est obtenu en faisant ingérer à l'animal un régime riche en graisse et en administrant un agent d'altération du foie au moins quatre fois.
PCT/JP2012/068878 2011-07-25 2012-07-25 Animal modèle de stéato-hépatite non alcoolique (nash) WO2013015331A1 (fr)

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WO2019088208A1 (fr) * 2017-11-01 2019-05-09 株式会社フェニックスバイオ Modèle de stéatohépatite non alcoolique humaine
JPWO2019088208A1 (ja) * 2017-11-01 2021-03-11 株式会社フェニックスバイオ ヒト非アルコール性脂肪性肝炎モデル
JP7233039B2 (ja) 2017-11-01 2023-03-06 株式会社フェニックスバイオ ヒト非アルコール性脂肪性肝炎モデル
WO2021043205A1 (fr) * 2019-09-03 2021-03-11 Crown Bioscience Inc. (Taicang) Procédés de génération de modèles animaux pour une stéatose hépatique non alcoolique
CN110652579A (zh) * 2019-11-12 2020-01-07 重庆医科大学附属第二医院 磷酸化胞壁酸酰二肽在制备构建爆发性肝损伤动物模型的药物中的应用
CN112088837A (zh) * 2020-09-22 2020-12-18 西北民族大学 一种非酒精性脂肪肝小鼠模型的构建方法

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