WO2019070024A1 - Animal model of nafld that can accompany fibrosis of liver, method for producing same, and feed for producing same - Google Patents

Animal model of nafld that can accompany fibrosis of liver, method for producing same, and feed for producing same Download PDF

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WO2019070024A1
WO2019070024A1 PCT/JP2018/037197 JP2018037197W WO2019070024A1 WO 2019070024 A1 WO2019070024 A1 WO 2019070024A1 JP 2018037197 W JP2018037197 W JP 2018037197W WO 2019070024 A1 WO2019070024 A1 WO 2019070024A1
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feed
liver
group
mice
nafld
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PCT/JP2018/037197
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French (fr)
Japanese (ja)
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真祐子 市村
勝久 大曲
幸一 常山
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林化成株式会社
国立大学法人奈良女子大学
長崎県公立大学法人
国立大学法人徳島大学
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Priority to JP2019547013A priority Critical patent/JP7126172B2/en
Publication of WO2019070024A1 publication Critical patent/WO2019070024A1/en

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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
    • 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
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/168—Steroids
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/50—Feeding-stuffs specially adapted for particular animals for rodents

Definitions

  • the present invention relates to a NAFLD model animal that may be associated with fibrosis of the liver, a method for producing the same, and a feed for producing the same.
  • Nonalcoholic fatty liver disease is a general term for liver disease with fat deposition excluding patients with no history of drinking, excluding those with obvious causes such as viral hepatitis, and histopathology
  • NASH Nonalcoholic steatohepatitis
  • NAFLD hepatocellular injury
  • hepatic fibrosis on the basis of relatively large hepatic steatosis and non-progressive disease state It is roughly divided into alcoholic fatty liver (NAFL).
  • Most patients with NAFLD have been complicated by obesity, diabetes, hyperinsulinemia, dyslipidemia and so on, and NAFLD and NASH patients are also increasing in Japan as the diet becomes westernized and lack of exercise, and the obese population increases. There is.
  • Patent Document 1 NASH model animals using KK-A y type 2 diabetes model mice are known.
  • An object of the present invention is to provide a convenient NAFLD model animal preparation technology that is more human-friendly and is made without using genetic modification technology.
  • the present invention relates to the following inventions.
  • a feed for producing a non-alcoholic fatty liver disease model mouse comprising cholic acid or a salt thereof and cholesterol.
  • a method for producing a non-alcoholic fatty liver disease model mouse comprising the step of feeding the mouse a feed according to any one of the above [1] to [3] and rearing the mouse.
  • a non-alcoholic fatty liver disease mouse model prepared by the method described in [4] above.
  • NAFLD model mice By using the feed of the present invention, NAFLD model mice can be produced.
  • the NAFLD model mouse of the present invention is particularly excellent in that it is a more convenient human NAFLD model animal, which is produced only by dietary administration without using genetic modification technology and is closer to humans.
  • the NAFLD model mouse of the present invention exhibits the NASH pathological condition or the NAFL pathological condition, and is further excellent in that it can more preferably show liver fibrosis. Liver fibrosis can develop in a relatively short time (eg, 9 weeks).
  • a preferable advantage of the NAFLD model mouse of the present invention for example, there is a large increase in weight of visceral fat per body weight or weight, and the point of exhibiting insulin resistance.
  • the NAFLD model mouse of the present invention is useful for elucidating the pathogenesis or mechanism of NAFLD, development of a novel prophylactic or therapeutic drug against NAFLD, particularly for screening of such prophylactic or therapeutic drug.
  • the feed of the present invention is also superior in that it does not have to be a nutrient-depleted feed such as methionine and choline-free feed (MCD).
  • FIG. 1 shows the results of serum cholesterol levels of each group after feeding at 9 weeks of age to 9 weeks.
  • FIG. 2 shows the results of serum alanine aminotransferase (ALT) values of each group after feeding at 9 weeks of age to 9 weeks.
  • FIG. 3 shows the results of liver triglyceride levels in each group after feeding at 9 weeks of age to 9 weeks.
  • FIG. 4 shows the results of liver histological findings of each group after feeding at 9 weeks of age to 9 weeks.
  • FIG. 5 shows the results of procollagen type I, alpha 1 (Col1a1) mRNA expression in each group after feeding for 9 weeks to 9 weeks.
  • FIG. 6 shows the results of transforming growth factor (Tgf) - ⁇ 1 mRNA expression in each group after feeding for 9 weeks to 9 weeks.
  • Tgf transforming growth factor
  • FIG. 7 shows the results of the total amount of energy intake for each group after feeding at 9 weeks of age to 9 weeks.
  • FIG. 8 shows the results of the final body weight of each group after feeding at 9 weeks of age to 9 weeks.
  • FIG. 9 shows the results of liver weight / body weight ratio of each group after feeding at 9 weeks of age to 9 weeks.
  • FIG. 10 shows the change in body weight of each group when the feed was taken from 9 weeks of age to 11 weeks.
  • FIG. 11 shows the change in body weight of each group when the feed was taken from 5 weeks of age to 15 weeks.
  • FIG. 12 shows the results of serum alanine aminotransferase (ALT) levels in each group after feeding at 6 weeks of age for 3 or 6 months.
  • FIG. ALT serum alanine aminotransferase
  • FIG. 13 shows the results of liver histological findings of each group after having been fed food for 6 or 7 months at 3 or 6 months.
  • FIG. 14 shows the results of procollagen type I, alpha 1 (Col1a1) mRNA expression in each group after feeding at 6 weeks of age for 3 or 6 months.
  • FIG. 15 shows the results of transforming growth factor (Tgf) - ⁇ 1 mRNA expression in each group after feeding at 6 weeks of age for 3 or 6 months.
  • FIG. 16 shows the results of liver weight / body weight ratio of each group after feeding at 6 weeks of age for 3 or 6 months.
  • FIG. 17 shows the change in body weight of each group after feed was taken from 6 weeks of age to 3 or 6 months.
  • FIG. 18 shows the results of liver histologic findings of each group after feed was ingested for 16 or 24 weeks from 5 or 9 weeks of age.
  • FIG. 19 shows the results of procollagen type I, alpha 1 (Col1a1) mRNA expression in each group after feed was ingested for 16 or 24 weeks from 5 or 9 weeks of age.
  • FIG. 20 shows the results of transforming growth factor (Tgf) - ⁇ 1 mRNA expression in each group after feed was ingested for 16 or 24 weeks from 5 or 9 weeks of age.
  • Tgf transforming growth factor
  • Feed The feed for producing the NAFLD model mouse of the present invention is characterized by containing cholic acid or a salt thereof and cholesterol.
  • Cholic acid or its salt cholic acid is a typical bile acid.
  • Cholic acid is produced in the liver by the metabolism of cholesterol, is sent to the gallbladder, is secreted in the duodenum, and is partially metabolized by microorganisms by microorganisms to become secondary bile acids such as deoxycholic acid.
  • Cholic acid is usually condensed with amino acids to form conjugated bile acids. Examples of amino acids include glycine and taurine.
  • the cholic acid may be glycocholic acid, which is a conjugated bile acid of cholic acid and glycine, or taurocholic acid, which is a conjugated bile acid of cholic acid and taurine.
  • the salt of cholic acid is preferably a pharmaceutically acceptable salt of cholic acid, and specifically, a salt with a strong base such as sodium salt or potassium salt, a salt with a weak base such as ammonia, etc. It can be mentioned. Cholic acid or a salt thereof can also be produced by known chemical synthesis methods, or commercially available products can be purchased and obtained. These all belong to the category of cholic acid used in the present invention.
  • the content of cholic acid or its salt in feed is not particularly limited as long as the effects of the present invention are not impaired, but it is usually 0.01 to 2% by mass, preferably 0.1 to 1% by mass, Preferably, it is less than 0.5% by mass (for example, 0.1 to 0.45% by mass), and particularly preferably 0.2 to 0.4% by mass. It is preferable that the content of cholic acid or its salt in feed is low (eg, more than 0% by mass and less than 2% by mass). The lower the amount of cholic acid, the better the NAFLD model mouse is obtained.
  • the mouse receiving the feed has a weight or an epididymal fat Etc. increase weight / weight of visceral fat or show insulin resistance.
  • the content of cholic acid or a salt thereof in feed is, for example, preferably 0.001% by mass or more and less than 0.5% by mass or 0.1% by mass or more and less than 0.4% by mass.
  • the content of cholic acid or its salt in feed tends to induce liver damage (hepatic fibrosis etc.) In some cases.
  • Cholesterol Cholesterol can also be produced by known chemical synthesis, microorganism culture, enzyme reaction and the like, and can also be obtained by purchasing commercially available products.
  • Examples of commercially available products include COLESTEROL NF (trade name) manufactured by Croda Japan Co., Ltd., cholesterol JSQI (trade name Japan Pharmacopoeia cholesterol) manufactured by Nippon Seika Co., Ltd., Nissui Marine Cholesterol manufactured by Japan Fisheries Co., Ltd. (Product Name) etc.
  • the cholesterol content in feed is not particularly limited as long as the effects of the present invention are not impaired, but it is usually 0.01 to 5% by mass, preferably 0.1 to 3% by mass, particularly preferably 1 to 1 .5 mass%.
  • the cholesterol content in the feed is preferably high (eg, 1% by mass or more).
  • the higher the cholesterol content in the feed the better the NAFLD model mice obtained.
  • the higher the cholesterol content in the feed the higher the feed intake, the higher the blood ALT value or the higher the degree of hepatic fibrosis.
  • the content of cholesterol in feed is, for example, preferably 0.01 to 2.5% by mass.
  • Standard Feed may be anything as long as it can be used for rearing mice, but it is usually a common ingredient (eg, water, crude protein, crude lipid, crude ash, crude fiber, soluble nitrogen-free) Or two or more of them (preferably all), vitamins (vitamin A, vitamin D3, vitamin E, vitamin K3, vitamin B1, vitamin B2, vitamin C, vitamin B6, inositol, biotin, pantothenic acid, niacin, choline , Folic acid, or a combination of two or more (preferably all) of them, minerals (calcium, phosphorus, magnesium, sodium, potassium, iron, aluminum, copper, zinc, cobalt, manganese, or two or more of them) (Preferably all combinations etc.), amino acids (isoleucine, leucine, lysine, methionine, cis) Emissions, containing phenylalanine, tyrosine, Suteonin, tryptophan, valine, arginine, histidine, a
  • Standard feeds examples include MF, MNF, CMF, CR-LPF, CRF-1 and the like manufactured by Oriental Yeast Co., Ltd., and CE-2 and the like manufactured by CLEA Japan, Inc. Among them, MF is preferable.
  • Standard diets can be reworded as laboratory animal diets that are commonly used in the art.
  • the standard feed content in the feed is not particularly limited as long as the effects of the present invention are not impaired, but it is usually 50 to 95% by mass, preferably 55 to 90% by mass, and particularly preferably 60 to 85% by mass. is there.
  • Lipid energy ratio The lipid energy ratio can be determined by the following equation.
  • Lipid energy ratio (%) ⁇ lipid amount (gram) x 9 kcal / total energy (kcal) ⁇ x 100
  • the lipid energy ratio of the feed is high (eg, 30% or more).
  • the higher the lipid energy ratio of the feed the better the NAFLD model mice obtained.
  • the lipid energy ratio of the feed is, for example, preferably 30 to 50%. Although it may be more than that, it is difficult to obtain a larger effect.
  • Fats and oils may be included in the feed.
  • the fats and oils are preferably fats and oils containing trans fatty acids or saturated fatty acids as constituent fatty acids.
  • fats and oils can be obtained by purchasing commercially available fats and oils. By feeding the diet containing these fats and oils to mice, excellent NAFLD model mice can be obtained.
  • feeding a feed containing these fats and oils to mice allows, for example, a NAFLD model mouse showing a high degree of fibrosis or increasing the weight of visceral fat per body weight or body weight to be obtained in a short period of time.
  • the content of fat and oil in feed is not particularly limited as long as the effects of the present invention are not impaired, but is usually 15 to 65% by mass, preferably 20 to 60% by mass, and particularly preferably 25 to 50% by mass. .
  • the feed of the present invention may be, if necessary, a feed for laboratory animals other than the above standard feed such as general feed, purified feed, sterile feed, specially formulated feed, etc .; preservative, antioxidant, stabilizer, antioxidant
  • any known additive commonly used in the field of feed such as pharmaceutical solvents, solubilizers, suspending agents, tonicity agents, etc .; and / or pharmacologically acceptable additives as required It can also be done.
  • these can be used alone or in combination of two or more depending on the intended feed form. A commercial item can be used for these.
  • the preservative is not particularly limited, and examples thereof include ethyl parahydroxybenzoate, chlorobutanol, benzyl alcohol, sodium dehydroacetate, sorbic acid and the like.
  • the antioxidant is not particularly limited, and examples thereof include sodium sulfite, ascorbic acid and the like.
  • the stabilizer is not particularly limited, and examples thereof include casein, casein sodium salt and the like.
  • Antioxidants include, for example, t-butylhydroquinone, butylhydroxyanisole, butylhydroxytoluene, and ⁇ -tocopherol, and derivatives thereof.
  • the feed may be processed into solid, liquid, emulsifier, paste, gel, powder, tablets, granules, pellets, capsules, syrups, suspensions or sticks.
  • solid it may be substantially cylindrical. These may be manufactured by known methods.
  • the feed of the present invention has a triglyceride accumulation action in the liver, an action to increase the ALT level in blood, and the like.
  • the feed of the present invention can cause the pathological condition of NAFLD (in particular, liver fibrosis) in mice receiving it.
  • the feed of the present invention can be produced by adding an appropriate amount of the above-mentioned components and kneading, stirring and / or mixing according to a known method or a method known per se.
  • the present invention provides a method for producing a non-alcoholic fatty liver disease mouse model, which comprises the step of feeding the feed of the present invention to a mouse for breeding.
  • the mouse is preferably not a genetically modified mouse.
  • a / J, C57BL / 6J, TSNO (Tsumura-Suzuki non-obese), TSOD (Tsumura-Suzuki obese diabetes) C57BL / 6N, C3H / HeN, C3H / HeJ, BALB / c, Inbred lines such as FVB / N, 129 + Ter / Sv, NOD / Shi, NC, FGS / Nga, CBA / JN, DBA / 1, DBA / 2, NC / Nga, AKR / J, etc.
  • a / J or TSNO is preferable among them.
  • C57BL / 6J is suitable as a standard model
  • a / J as an Asian model or lean model
  • TSNO as a strong fibrosis model.
  • Asians are reported to become NAFLD even if they are not obese, so A / J showing obesity resistance is suitable as an Asian model or lean model.
  • TSNO more particularly, it is supported by Test Example 4 described below that it is suitable as a diet-induced hepatic fibrosis model.
  • the mouse freely feeds the feed by placing the feed in a mouse cage or in a cage lid (reticular) according to a conventional method.
  • the daily food intake of a mouse is usually about 3 to 5 grams, but is not limited thereto, and may be an amount that a mouse individual can actually eat under breeding conditions.
  • Feeding period is not particularly limited as long as feeding is started after weaning, but for example, it is preferably 4 weeks or more, and more preferably 5-10 weeks.
  • the feed period is, for example, preferably 6 weeks or more, more preferably 9 weeks or more, more preferably 10 weeks or more, and still more preferably 11 weeks or more.
  • the longer the feeding period eg, 9 weeks or more
  • the feed period is preferably 6 to 52 weeks, more preferably 10 to 26 weeks or 26 to 52 weeks.
  • the feed period is preferably as long as the amount of cholic acid is small. The lower the cholesterol, the longer the feed period.
  • the feeding period is longer as the lipid energy ratio is lower.
  • the breeding environment of the mouse is preferably a room with a temperature of 20 to 25 ° C., a humidity of 50 to 60%, a 12 hour light period, and a 12 hour dark cycle.
  • NAFLD model animal Non-alcoholic fatty liver disease (NAFLD) is a non-alcoholic steatohepatitis with hepatocellular injury (balloon-like change) and hepatic fibrosis, based on histopathologically large hepatic steatosis. (NASH) and non-alcoholic fatty liver (NAFL), in which progression of pathological condition is not observed.
  • the NAFLD model mouse of the present invention is particularly excellent in that it is a human-friendly, highly convenient NAFLD model animal that is produced without using genetic modification technology. Mice have different properties compared to other experimental animals (eg, rats etc.). For example, mice are closer to humans in having a gallbladder.
  • the mouse is highly convenient, for example, in that the size of the individual is small, the amount of food can be small, and it is easy to handle.
  • the NAFLD model mouse of the present invention shows the pathological condition of NAFLD, for example, blood triglyceride level, liver triglyceride level, blood cholesterol level, blood glucose level, blood ALT level by a known biochemical method per se. The level, insulin level in blood, leptin level in blood, etc. can be measured, and it can be easily confirmed whether or not the behavior similar to that of human NAFLD is exhibited.
  • Similar behavior is, for example, blood triglyceride level, liver triglyceride level, blood cholesterol level, liver cholesterol level, blood glucose level, blood glucose level in the non-diseased group (group not receiving the feed of the present invention) It is said that the tendency (decrease or increase) of these values in comparison with ALT value, blood insulin value, insulin resistance index, blood leptin value etc. is the same.
  • any one or more of blood insulin level, blood glucose level, blood cholesterol level, blood ALT level and liver triglyceride level is significantly increased as compared to the group not receiving the feed of the present invention.
  • the group to which the feed of the present invention has been administered is useful as a NAFLD model mouse.
  • the group receiving the feed of the present invention is NAFLD
  • the group receiving the feed of the present invention is superior as a NAFLD model mouse.
  • the fact that the NAFLD model mouse of the present invention shows NASH disease states that liver tissue is stained by, for example, hematoxylin and eosin (HE) staining or azan (Azan) staining, and the "NASH Clinical Research Network Scoring System".
  • liver tissue is pathologically observed, and 0 to 2 points are not NASH, 3 to 4 points are judged pending, 5 points out of 8 points in the total points of NAFLD activity score (NAS) shown in Table 1 below. The above can be judged as NASH.
  • NAS NAFLD activity score
  • the NAFLD model mouse of the present invention may more preferably exhibit hepatic fibrosis as one of NASH pathological conditions. Above all, adopting TSNO mice as mice is preferable in that fibrosis of the liver can be shown. Assessment of liver fibrosis can be performed, for example, according to the criteria shown in Table 2 below, by pathologically observing liver tissue.
  • the fact that the NAFLD model mouse of the present invention exhibits the NAFL pathological condition can be confirmed, for example, by recognizing fatty liver by tissue diagnosis and not recognizing hepatocellular injury (balloon-like degeneration) or fibrosis.
  • the present invention includes embodiments in which the above-described configurations are variously combined within the technical scope of the present invention as long as the effects of the present invention can be obtained.
  • Test Example 1 Animal model and experimental design 15 8-week-old male C57BL / 6J mice (B6 mice) and 15 8-week-old male A / J mice (all from Japan SLC, Shizuoka) were purchased, and the first week was Standard feed (MF; Oriental Yeast, Tokyo) was ingested to acclimate to the breeding environment.
  • MF Standard feed
  • Oriental Yeast Tokyo
  • B6 mice and A / J mice are normal group (group N) fed MF as a standard diet from 9 weeks of age after acclimatization, 28.75% by mass of powdered palm oil in MF, 1.25 mass of cholesterol % And low cholesterol group (LC group) fed a high-fat diet supplemented with 0.5% by weight of sodium cholate, MF 27.5% by weight of palm oil, 2.5% by weight of cholesterol and cholate sodium salt
  • the high-cholesterol group (HC group) which received a high-fat diet to which 0.5% by mass had been added was divided into 4 to 6 animals each for 3 groups and reared until 18 weeks of age.
  • the feed was purchased from Oriental yeast for preparation.
  • the respective feed composition and energy are shown in Table 3 below.
  • Total cholesterol in serum Measurement of total cholesterol in serum was carried out according to the method described in the instruction using Cholesterol E-Test Wako (Wako Pure Chemical Industries), which is a measurement kit using the cholesterol oxidase / DAOS method. The measurement was performed twice for one sample, and the average value was measured.
  • ALT in serum Using the transaminase C-II Test Wako (Wako Pure Chemical Industries, Ltd.), which is a measurement kit using the (POP ⁇ TOOS) method, the serum ALT concentration was measured according to the method described in the instruction. The measurement was performed twice for one sample, and the average value was taken as the measurement value.
  • Liver triglyceride Liver lipids were extracted using 0.5 g of cryopreserved liver, and the TG concentration was measured. Extraction was performed by the method of Folch et al. 0.5 g of liver tissue is homogenized, transferred while transferring to a measuring flask with a methanol / chloroform mixed solution (volume ratio 1: 2) with about 50 mL, shaken at 40 ° C. for 30 minutes, returned to room temperature and mixed with methanol / chloroform The solution was added to 50 mL. After filtration, about 9 mL of distilled water was added and mixed gently, and the aqueous layer was discarded.
  • ⁇ Measurement method> After returning to room temperature, the sample was taken in a test tube, and from the sample diluted with hexane, another sample was taken in a test tube and dried in nitrogen, and then 100 ⁇ L of isopropanol was added and mixed well. This was used for the measurement. Triglyceride E-Test Wako (Wako Pure Chemical Industries) was used for the measurement.
  • liver tissue staining The paraffin block in which the liver tissue was embedded was sliced with a microsome to a thickness of 4 ⁇ m, deparaffinized and washed with water, and then stained for 5 minutes with Mayer's hematoxylin solution. After washing with water, it was stained with eosin solution for 2 to 3 minutes, fractionated with alcohol, and then dehydrated, cleared and sealed.
  • a paraffin block embedded with liver tissue is sliced with a microtome to a thickness of 4 ⁇ m, deparaffinized and washed with water, and then 10% (w / v) aqueous potassium dichromate solution and 10% (w / v) trily It was immersed for 10 to 20 minutes in a mordant mixed with an equal amount of aqueous solution of chloroacetic acid. After washing again with water, it was immersed in azocarmine G solution at room temperature for 30 minutes or more. It was washed with distilled water, separated with aniline and alcohol for several seconds, stopped with acetic acid and alcohol, washed with water, and microscopically checked to confirm the dyed state.
  • NAFLD activity score (NAS)
  • 0 to 2 points are not NASH out of a total of 8 points, 3 to 4 points are judged Withheld, it was judged that NASH was more than 5 points.
  • evaluation of fibrosis was also performed.
  • Liver fibrosis marker (Col1a1, Tgf- ⁇ 1)
  • Total RNA was extracted from cryopreserved liver tissue using RNAiso Plus (Takara Bio, Shiga) according to the method described in the manual.
  • Reverse transcription reaction was performed using total RNA and ReverTra Ace qPCR RT Master Mix (Toyobo, Osaka), and cDNA was prepared and used for Real-time PCR.
  • PCR is carried out using LightCycler Nano (Roche Diagnostics, Tokyo, Japan) or STEP One real-time PCR system (Applied Biosystems, Carlsbad, CA, USA), and THUNDERBIRD SYBR qPCR is used to prepare a PCR reaction solution. Mix (Toyobo) was used.
  • Primer synthesis was requested by FASMAC (Kanagawa) and purchased. The PCR reaction was carried out with a standard program as described. The relative ratio of the expression level of each target gene (Col1a1, Tgf- ⁇ 1) was analyzed by the comparative Ct method using the ⁇ -actin gene as an internal standard.
  • a group receiving a normal diet is B6 N
  • a group receiving a high fat diet supplemented with 1.25 mass% cholesterol is a B6 LC group
  • a group receiving a high fat diet supplemented with 2.5 mass% cholesterol a B6 HC group write.
  • a / J mice are also referred to as A / JN group, A / JLC group, and A / JHC group.
  • FIG. 1 shows serum cholesterol level
  • FIG. 2 shows serum ALT level
  • FIG. 3 shows liver TG level.
  • the intake of the feed of the present invention significantly increased the amount of triglyceride in the liver (FIG. 3).
  • intake of the feed of the present invention significantly increased serum cholesterol and ALT levels of B6 mice and A / J mice (FIGS. 1 and 2).
  • Liver histopathology of B6LC group, B6HC group, A / JLC group, and A / JHC group showed individuals with NAFLD activity score of 5 or more (Table 4 and Figure 4), and individuals diagnosed with NASH were obtained It was done. There are more mice with higher NAFLD activity score in A / J mice than in B6 mice, A / J mice have NASH susceptibility, or A / J mice have sensitivity to feed components used in this experiment. It was confirmed to be high.
  • liver fibrosis The presence of liver fibrosis findings is a lesion that should be noted as there has been a report of its prognostic deterioration.
  • moderate fibrotic lesions at stage 2 were identified (FIG. 4).
  • Fibrosis showed cholesterol-dependent deterioration in both B6 and A / J mice.
  • a / J mice showed higher fibrotic lesions as compared to B6 mice.
  • Cholesterol accumulation in the liver in A / J mice is at a higher level than in B6 mice, which may be responsible for high liver fibrosis in A / J mice.
  • liver fibrosis marker The expression level of each mRNA (Col1a1, Tgf- ⁇ 1) which is a liver fibrosis marker shows a significant increase or a tendency to increase in LC group and HC group of B6 mouse and A / J mouse in comparison with each normal group ( Figures 5 and 6).
  • Test example 2 (1) Experimental method Twenty-eight 8-week-old male C57BL / 6J mice (Japan SLC) were purchased, and a standard diet (MF; Oriental yeast) was ingested for the first week to acclimate to the breeding environment. Normal food group fed MF, which is a standard feed, from 9 weeks of age after acclimatization, to 25% by mass of powdered palm oil (trade name: powdered oil and fat P-80; manufactured by Nagasesan Bio), and cholesterol (trade name: MF) Cholesterol: 0.2% cholic acid ingested feed containing 1.25% by weight Wako Pure Chemical Industries, Ltd.
  • MF powdered palm oil
  • MF cholesterol
  • Cholesterol 0.2% cholic acid ingested feed containing 1.25% by weight Wako Pure Chemical Industries, Ltd.
  • cholic acid sodium salt (trade name sodium cholate; Wako Pure Chemical Industries) 0.2% by weight
  • Added food group 3 to 3 each of 3 groups of 3 groups of cholic acid 0.4% added food group to which 25% by mass powdered palm oil, 1.25% by mass cholesterol and 0.4% by mass cholate were added to MF Separated and kept until 20 weeks old. Food and water were free intake, and body weight was measured once a week during the breeding period. All animals were kept in a room set at a temperature of 24 ° C., a humidity of 55%, a light period of 7-19 o'clock and a dark cycle of 19-7 o'clock. The present animal experiment was conducted in accordance with "Guideline on animal experiment of Nara Women's University" and "Standard for breeding and storage of experimental animal (announcement No. 6 of the Prime Minister's notice in March 1959)".
  • Test Example 3 (1) Experimental method Twenty-four 4-week-old male C57BL / 6J mice (Japan SLC) were purchased, and a standard diet (MF; Oriental yeast) was ingested for the first week to acclimate to the breeding environment.
  • MF standard diet
  • Normal food group fed MF which is a standard feed, from 5 weeks of age after acclimatization, 25% by mass powdered palm oil (trade name: Powdered oil and fat P-80; manufactured by Nagasesan Bio) in MF, cholesterol (trade name) Cholesterol: 0.1% cholic acid ingested feed supplemented with 1.25% by mass Wako Pure Chemical Industries, Ltd.
  • Test Example 4 (1) Experimental method Twenty six-week-old male Tsumura-Suzuki non-obese (TSNO) mice and 12 Tsumura-Suzuki obese diabetes (TSOD) mice (all at the Animal Reproductive Research Institute) were obtained, and a standard diet (MF; Oriental yeast), 28.75 wt% powdered palm oil, 1.25 wt% cholesterol and 0.5 wt% cholate sodium salt added to MF It was divided into fat and cholesterol diet (HFC) groups. The animals were sacrificed at 3 months and 6 months after the start of feed intake. Each group had 6 animals, and food and water were freely available. The experiment was commissioned to the Animal Breeding Research Institute, and the feed was requested to be produced by Oriental yeast.
  • MF standard diet
  • HFC fat and cholesterol diet
  • ALT in serum liver histology findings, liver fibrosis marker (Col1a1) by the method described in the column of serum / liver biochemical measurement, histopathological examination, and liver fibrosis marker described in Test Example 1 , Tgf- ⁇ 1).
  • the relative ratio of the expression level of each target gene (Col1a1, Tgf- ⁇ 1) was analyzed by the comparative Ct method using a glyceroldede 3-phosphate dehydrogenase (Gapdh) gene as an internal standard.
  • weight measurement was performed once every two or four weeks.
  • the analysis results of each item are shown as mean value ⁇ standard error (SE). Tests for significant differences between groups are performed by one-way analysis of variance and Bonferroni's method (multiple comparisons) using the statistical analysis software GraphPad Prism 7.02, and significant differences P ⁇ 0.05 are statistically significant. I judged that there was.
  • FIG. 12 shows the results of serum ALT levels. As apparent from FIG. 12, the intake of the feed of the present invention for 3 months or 6 months significantly increased the serum ALT value of TSNO and TSOD mice.
  • FIG. 13 shows the results of liver histological findings.
  • NASH onset and further fibrotic lesions in TSNO mice were confirmed by intake for 3 or 6 months of the feed of the present invention.
  • mild steatosis, inflammation and balloon-like hepatocytes were observed in the HFC group and NASH was developed.
  • fibrotic lesions were observed in stages 1 to 2 in the HFC-TSNO-3m group and in stages 2 to 3 in the HFC-TSNO-6m group.
  • fibrotic lesions were about 0-1 stage in the HFC-TSOD-3m group and about 1-2 stages in the HFC-TSNO-6m group.
  • TSNO mice rather than TSOD mice in terms of highly inducing liver fibrosis.
  • this model has a fairly high degree of fibrosis and can be said to be an excellent model animal to develop advanced NAFLD.
  • FIGS. 14 and 15 show the results of liver fibrosis markers. As apparent from FIGS. 14 and 15, a significant increase in liver fibrosis marker in TSNO mice was confirmed by the feed intake of the present invention. Specifically, the expression level of each mRNA (Col1a1, Tgf- ⁇ 1) in the liver was significantly increased in the HFC group of TSNO mice as compared to each NM group.
  • FIG. 16 shows the result of liver weight / body weight ratio
  • FIG. 17 shows the transition of weight.
  • a significant increase in liver weight / body weight ratio of TSNO and TSOD mice was confirmed by the feed intake of the present invention. More specifically, in the HFC-TSNO group, the liver weight / body weight ratio showed a high value as compared with the same age NM-TSNO and HFC-TSOD groups. There was no significant difference in body weight between NM-TSNO-3m and HFC-TSNO-3m (FIG. 17).
  • Test Example 5 (1) Experimental Method Twenty-six male 4- and 8-week-old male C57BL / 6J mice (Japan SLC) were purchased, and a standard diet (MF; Oriental yeast) was ingested for the first week to acclimate to the breeding environment. A normal diet group ingested MF, which is a standard diet, from 5 and 9 weeks of age after acclimatization, 25% by mass of powdered palm oil (trade name Powdered oil and fat P-80; manufactured by Nagasesan Bio) in MF, cholesterol (MF) Trade name: cholesterol; Wako Pure Chemical Industries, Ltd.
  • MF powdered palm oil
  • MF cholesterol
  • Cholic acid sodium salt (trade name: sodium cholate; Wako Pure Chemical Industries, Ltd.) 0.1% by mass Cholic acid ingested with feed added thereto.
  • 1% added food group 25% by mass powdered palm oil, 1.25% by mass cholesterol and 0.2% by mass cholic acid sodium salt added to caffeic acid 0.2% added food group with MF, powdered palm oil 25 to MF
  • MF powdered palm oil 25 to MF
  • Each group was divided into 3 to 5 animals each in 4 groups of the cholic acid 0.4% -added food group to which mass%, cholesterol 1.25% by mass, and cholic acid sodium salt 0.4% by mass were added, and reared for 16 or 24 weeks. Food and water were free intake, and body weight was measured once a week during the breeding period.
  • the animals were fasted for 8 hours before sacrifice, then underwent laparotomy under isoflurane inhalation anesthesia, and blood was collected from the heart using a syringe. Immediately, the liver and peritesticular fat were removed, and after washing with 0.9% (w / v) saline, the weight was measured. A part of the liver was excised and fixed with 10% (w / v) neutral formalin solution.
  • glucose glucose level Using glucose CII-Test Wako (Wako Pure Chemical Industries), which is a measurement kit using the mutarotase-GOD method, glucose in serum was measured according to the method described in the instruction. The measurement was performed twice for one sample, and the average value was measured.
  • Serum insulin level Using the mouse insulin measurement kit (Morinaga Institute of Science), which is a measurement kit using the ELISA method, insulin in serum was measured according to the method described in the instruction. The measurement was performed twice for one sample, and the average value was measured.
  • Insulin resistance index serum insulin level (ng / mL) ⁇ serum glucose level (mg / dL) 405 405
  • Table 5 shows the results of serum ALT level, serum glucose level, serum insulin level and insulin resistance index.
  • the feed intake of the present invention significantly increased the serum ALT value of C57BL / 6J mice.
  • the serum ALT value was higher than that of the cholic acid 0.2% -added food group.
  • the insulin resistance index tended to be higher compared to the cholic acid 0.4% -added food group. Since insulin resistance is frequently observed in NASH disease states in humans, it is more desirable that the insulin resistance index shows a higher value than the normal food intake group, and the cholic acid addition concentration is 0.2% than 0.4%. It can be said that a better NAFLD model animal can be produced.
  • FIG. 18 shows the results of liver histological findings.
  • hepatic fibrosis in C57BL / 6J mice was confirmed by the feed intake of the present invention.
  • steatosis and severe inflammation were observed in the cholic acid 0.4% -added diet group.
  • fibrosis was very mild around the central vein. Mild to moderate steatosis and inflammation were seen in the 0.2% and 0.1% chow diet diet groups, with characteristic findings of NASH.
  • Fibrosis was a mild lesion (stage 1) around the central vein and around hepatocytes.
  • FIGS. 19 and 20 show the results of liver fibrosis markers. As apparent from FIGS. 19 and 20, a significant increase or a tendency of a marker for fibrosis of liver of C57BL / 6J mice by the feed intake of the present invention was confirmed.
  • Table 6 shows the results of body weight and testis surrounding fat weight / body weight ratio.
  • the body weight and testicular fat weight / body weight in comparison with the same-week-old normal diet group An increase in the ratio was confirmed. That is, it was confirmed that a better NAFLD model mouse can be obtained by feeding a feed having a cholic acid addition concentration of less than 0.4% by mass (for example, 0.1 or 0.2% by mass).
  • the model animal of the present invention a method for producing the same, and a feed for producing the same are useful for elucidating the pathogenesis or mechanism of NAFLD, development of a novel prophylactic or therapeutic drug for NAFLD, and the like.

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Abstract

The present invention addresses the problem of providing a technique for producing an animal model of NAFLD, the animal being produced without the use of genetic modification, being closer to humans, and being highly convenient. The present invention provides: a feed for producing a mouse model of non-alcoholic fatty liver disease, the feed containing cholic acid or a salt thereof, and also containing cholesterol; and a method for producing a mouse model of non-alcoholic fatty liver disease, the method being characterized by including a step for feeding a mouse said feed.

Description

肝の線維化を伴い得るＮＡＦＬＤモデル動物、その作製方法、及びそれを作製するための飼料NAFLD model animal which may be associated with fibrosis of liver, method for producing the same, and feed for producing the same
　本発明は、肝の線維化を伴い得るＮＡＦＬＤモデル動物、その作製方法、及びそれを作製するための飼料に関する。 The present invention relates to a NAFLD model animal that may be associated with fibrosis of the liver, a method for producing the same, and a feed for producing the same.
　非アルコール性脂肪肝疾患（ｎｏｎａｌｃｏｈｏｌｉｃ　ｆａｔｔｙ　ｌｉｖｅｒ　ｄｉｓｅａｓｅ：ＮＡＦＬＤ）は、飲酒歴がない患者の、ウィルス性肝炎などの原因が明らかなものを除外した脂肪沈着を伴う肝疾患の総称であり、病理組織学的に大滴性の肝脂肪変性を基本として、肝細胞障害（風船様の変化）や肝線維化がみられる非アルコール性脂肪肝炎（ｎｏｎａｌｃｏｈｏｌｉｃ　ｓｔｅａｔｏｈｅｐａｔｉｔｉｓ：ＮＡＳＨ）と、病態の進行がみられない非アルコール性脂肪肝（ｎｏｎａｌｃｏｈｏｌｉｃ　ｆａｔｔｙ　ｌｉｖｅｒ：ＮＡＦＬ）に大別される。ＮＡＦＬＤ患者の多くは、肥満や糖尿病、高インスリン血症、脂質異常症などを合併しており、食生活の欧米化や運動不足、肥満人口の増加に伴いわが国でもＮＡＦＬＤやＮＡＳＨ患者が増加してきている。 Nonalcoholic fatty liver disease (NAFLD) is a general term for liver disease with fat deposition excluding patients with no history of drinking, excluding those with obvious causes such as viral hepatitis, and histopathology Nonalcoholic steatohepatitis (NASH) with hepatocellular injury (balloon-like changes) and hepatic fibrosis on the basis of relatively large hepatic steatosis and non-progressive disease state It is roughly divided into alcoholic fatty liver (NAFL). Most patients with NAFLD have been complicated by obesity, diabetes, hyperinsulinemia, dyslipidemia and so on, and NAFLD and NASH patients are also increasing in Japan as the diet becomes westernized and lack of exercise, and the obese population increases. There is.
　これまでに、ＫＫ－Ａｙ２型糖尿病モデルマウスを用いたＮＡＳＨモデル動物が知られている（特許文献１）。 So far, NASH model animals using KK-A y type 2 diabetes model mice are known (Patent Document 1).
特開２０１５－１８６４８４号公報JP, 2015-186484, A
　本発明は、遺伝子改変技術を使用しないで作製され、よりヒトに近く、利便性が高いＮＡＦＬＤモデル動物作成技術を提供することを目的とする。 An object of the present invention is to provide a convenient NAFLD model animal preparation technology that is more human-friendly and is made without using genetic modification technology.
　本発明は以下の発明に関する。
［１］コール酸又はその塩、及びコレステロールを含有することを特徴とする非アルコ－ル性脂肪肝疾患モデルマウス作製用飼料。
［２］標準飼料を含有することを特徴とする前記［１］記載の飼料。
［３］脂質エネルギー比率が３０％以上であることを特徴とする前記［１］又は［２］に記載の飼料。
［４］マウスに前記［１］～［３］のいずれかに記載の飼料を与えて飼育する工程を含むことを特徴とする非アルコ－ル性脂肪肝疾患モデルマウスの作製方法。
［５］前記［４］に記載の方法により作製された非アルコ－ル性脂肪肝疾患モデルマウス。 The present invention relates to the following inventions.
[1] A feed for producing a non-alcoholic fatty liver disease model mouse comprising cholic acid or a salt thereof and cholesterol.
[2] The feed according to the above [1], which contains a standard feed.
[3] The feed according to [1] or [2], wherein a lipid energy ratio is 30% or more.
[4] A method for producing a non-alcoholic fatty liver disease model mouse, comprising the step of feeding the mouse a feed according to any one of the above [1] to [3] and rearing the mouse.
[5] A non-alcoholic fatty liver disease mouse model prepared by the method described in [4] above.
　本発明の飼料を用いることにより、ＮＡＦＬＤモデルマウスを作製することができる。本発明のＮＡＦＬＤモデルマウスは、遺伝子改変技術を使用しないで食餌投与のみで作製され、よりヒトに近く、利便性が高いＮＡＦＬＤモデル動物である点において、特に優れている。また、本発明のＮＡＦＬＤモデルマウスは、ＮＡＳＨ病態又はＮＡＦＬ病態を示し、より好ましくはさらに肝の線維化を示し得る点でも優れている。肝の線維化は比較的短時間（例えば９週間）で発症し得る。また、本発明のＮＡＦＬＤモデルマウスの好ましい利点として、例えば、体重又は体重当たりの内臓脂肪の重さの増加幅が大きい点、インスリン抵抗性を呈する点が挙げられる。本発明のＮＡＦＬＤモデルマウスは、ＮＡＦＬＤの病態又はメカニズムの解明、ＮＡＦＬＤに対する新規予防又は治療薬の開発、特にかかる予防又は治療薬のスクリーニング等に有用である。
　さらには、本発明の飼料は、メチオニン及びコリン無添加飼料（ＭＣＤ）のような栄養素を欠乏させていない飼料でなくてもよい点でも優れている。 By using the feed of the present invention, NAFLD model mice can be produced. The NAFLD model mouse of the present invention is particularly excellent in that it is a more convenient human NAFLD model animal, which is produced only by dietary administration without using genetic modification technology and is closer to humans. In addition, the NAFLD model mouse of the present invention exhibits the NASH pathological condition or the NAFL pathological condition, and is further excellent in that it can more preferably show liver fibrosis. Liver fibrosis can develop in a relatively short time (eg, 9 weeks). In addition, as a preferable advantage of the NAFLD model mouse of the present invention, for example, there is a large increase in weight of visceral fat per body weight or weight, and the point of exhibiting insulin resistance. The NAFLD model mouse of the present invention is useful for elucidating the pathogenesis or mechanism of NAFLD, development of a novel prophylactic or therapeutic drug against NAFLD, particularly for screening of such prophylactic or therapeutic drug.
Furthermore, the feed of the present invention is also superior in that it does not have to be a nutrient-depleted feed such as methionine and choline-free feed (MCD).
図１は、９週齢時から９週間飼料を摂取させた後の、各群の血清コレステロール値の結果を示す。FIG. 1 shows the results of serum cholesterol levels of each group after feeding at 9 weeks of age to 9 weeks. 図２は、９週齢時から９週間飼料を摂取させた後の、各群の血清アラニンアミノトランスフェラーゼ（ＡＬＴ）値の結果を示す。FIG. 2 shows the results of serum alanine aminotransferase (ALT) values of each group after feeding at 9 weeks of age to 9 weeks. 図３は、９週齢時から９週間飼料を摂取させた後の、各群の肝臓トリグリセリド値の結果を示す。FIG. 3 shows the results of liver triglyceride levels in each group after feeding at 9 weeks of age to 9 weeks. 図４は、９週齢時から９週間飼料を摂取させた後の、各群の肝組織学的所見の結果を示す。FIG. 4 shows the results of liver histological findings of each group after feeding at 9 weeks of age to 9 weeks. 図５は、９週齢時から９週間飼料を摂取させた後の、各群のprocollagen type I, alpha 1（Ｃｏｌ１ａ１） ｍＲＮＡ発現の結果を示す。FIG. 5 shows the results of procollagen type I, alpha 1 (Col1a1) mRNA expression in each group after feeding for 9 weeks to 9 weeks. 図６は、９週齢時から９週間飼料を摂取させた後の、各群のtransforming growth factor(Ｔｇｆ)－β１ｍＲＮＡ発現の結果を示す。FIG. 6 shows the results of transforming growth factor (Tgf) -β1 mRNA expression in each group after feeding for 9 weeks to 9 weeks. 図７は、９週齢時から９週間飼料を摂取させた後の、各群の総摂取エネルギー量の結果を示す。FIG. 7 shows the results of the total amount of energy intake for each group after feeding at 9 weeks of age to 9 weeks. 図８は、９週齢時から９週間飼料を摂取させた後の、各群の終体重の結果を示す。FIG. 8 shows the results of the final body weight of each group after feeding at 9 weeks of age to 9 weeks. 図９は、９週齢時から９週間飼料を摂取させた後の、各群の肝臓重量／体重比の結果を示す。FIG. 9 shows the results of liver weight / body weight ratio of each group after feeding at 9 weeks of age to 9 weeks. 図１０は、９週齢時から１１週間飼料を摂取させた場合の各群の体重の推移を示す。FIG. 10 shows the change in body weight of each group when the feed was taken from 9 weeks of age to 11 weeks. 図１１は、５週齢時から１５週間飼料を摂取させた場合の各群の体重の推移を示す。FIG. 11 shows the change in body weight of each group when the feed was taken from 5 weeks of age to 15 weeks. 図１２は、６週齢時から３又は６ヶ月間飼料を摂取させた後の、各群の血清アラニンアミノトランスフェラーゼ（ＡＬＴ）値の結果を示す。FIG. 12 shows the results of serum alanine aminotransferase (ALT) levels in each group after feeding at 6 weeks of age for 3 or 6 months. 図１３は、６週齢時から３又は６ヶ月間飼料を摂取させた後の、各群の肝組織学的所見の結果を示す。FIG. 13 shows the results of liver histological findings of each group after having been fed food for 6 or 7 months at 3 or 6 months. 図１４は、６週齢時から３又は６ヶ月間飼料を摂取させた後の、各群のprocollagen type I, alpha 1（Ｃｏｌ１ａ１） ｍＲＮＡ発現の結果を示す。FIG. 14 shows the results of procollagen type I, alpha 1 (Col1a1) mRNA expression in each group after feeding at 6 weeks of age for 3 or 6 months. 図１５は、６週齢時から３又は６ヶ月間飼料を摂取させた後の、各群のtransforming growth factor(Ｔｇｆ)－β１ｍＲＮＡ発現の結果を示す。FIG. 15 shows the results of transforming growth factor (Tgf) -β1 mRNA expression in each group after feeding at 6 weeks of age for 3 or 6 months. 図１６は、６週齢時から３又は６ヶ月間飼料を摂取させた後の、各群の肝臓重量／体重比の結果を示す。FIG. 16 shows the results of liver weight / body weight ratio of each group after feeding at 6 weeks of age for 3 or 6 months. 図１７は、６週齢時から３又は６ヶ月間飼料を摂取させた後の、各群の体重の推移を示す。FIG. 17 shows the change in body weight of each group after feed was taken from 6 weeks of age to 3 or 6 months. 図１８は、５又は９週齢時から１６又は２４週間飼料を摂取させた後の、各群の肝組織学的所見の結果を示す。FIG. 18 shows the results of liver histologic findings of each group after feed was ingested for 16 or 24 weeks from 5 or 9 weeks of age. 図１９は、５又は９週齢時から１６又は２４週間飼料を摂取させた後の、各群のprocollagen type I, alpha 1（Ｃｏｌ１ａ１） ｍＲＮＡ発現の結果を示す。FIG. 19 shows the results of procollagen type I, alpha 1 (Col1a1) mRNA expression in each group after feed was ingested for 16 or 24 weeks from 5 or 9 weeks of age. 図２０は、５又は９週齢時から１６又は２４週間飼料を摂取させた後の、各群のtransforming growth factor(Ｔｇｆ)－β１ｍＲＮＡ発現の結果を示す。FIG. 20 shows the results of transforming growth factor (Tgf) -β1 mRNA expression in each group after feed was ingested for 16 or 24 weeks from 5 or 9 weeks of age.
１．飼料
　本発明のＮＡＦＬＤモデルマウス作製用飼料は、コール酸又はその塩、及びコレステロールを含有することを特徴とする。 1. Feed The feed for producing the NAFLD model mouse of the present invention is characterized by containing cholic acid or a salt thereof and cholesterol.
〔飼料含有成分〕
　コール酸又はその塩
　コール酸は、代表的な胆汁酸である。コール酸はコレステロールの代謝によって肝臓で作られ、胆嚢に送られ、十二指腸に分泌されて、その一部が微生物によって代謝され、デオキシコール酸等の二次胆汁酸となる。コール酸は、通常アミノ酸と縮合して抱合胆汁酸となって存在している。アミノ酸として、グリシン又はタウリン等が挙げられる。コール酸は、コール酸とグリシンとの抱合胆汁酸であるグリココール酸、又はコール酸とタウリンとの抱合胆汁酸であるタウロコール酸であってもよい。
　コール酸の塩は、コール酸の薬学的に許容される塩であることが好ましく、具体的には、ナトリウム塩、カリウム塩等の強塩基との塩、アンモニア等の弱塩基との塩等が挙げられる。コール酸又はその塩は、公知の化学合成法により製造することもでき、また市販品を購入して入手することもできる。これらは全て、本発明で使用されるコール酸の範疇に属する。 [Feed-containing ingredients]
Cholic acid or its salt cholic acid is a typical bile acid. Cholic acid is produced in the liver by the metabolism of cholesterol, is sent to the gallbladder, is secreted in the duodenum, and is partially metabolized by microorganisms by microorganisms to become secondary bile acids such as deoxycholic acid. Cholic acid is usually condensed with amino acids to form conjugated bile acids. Examples of amino acids include glycine and taurine. The cholic acid may be glycocholic acid, which is a conjugated bile acid of cholic acid and glycine, or taurocholic acid, which is a conjugated bile acid of cholic acid and taurine.
The salt of cholic acid is preferably a pharmaceutically acceptable salt of cholic acid, and specifically, a salt with a strong base such as sodium salt or potassium salt, a salt with a weak base such as ammonia, etc. It can be mentioned. Cholic acid or a salt thereof can also be produced by known chemical synthesis methods, or commercially available products can be purchased and obtained. These all belong to the category of cholic acid used in the present invention.
　飼料中のコール酸又はその塩の含有量は、本発明の効果を妨げない限り特に限定されないが、通常０．０１～２質量％であり、好ましくは０．１～１質量％であり、より好ましくは０．５質量％未満（例えば０．１～０．４５質量％）であり、特に好ましくは０．２～０．４質量％である。
　飼料中のコール酸又はその塩の含有量が少ないことが好ましい（例えば、０質量％超２質量％未満）。コール酸量が少ないほど、より優れたＮＡＦＬＤモデルマウスが得られる。具体的には、例えば、飼料中のコール酸又はその塩の含有量が少ない（例えば０．５質量％未満又は０．４質量％未満）ほど、飼料を摂取したマウスは、体重又は副睾丸脂肪等の内臓脂肪の重さ／体重が増加し、又はインスリン抵抗性を示す。飼料中のコール酸又はその塩の含有量は、具体的には、例えば０．００１質量％以上０．５質量％未満又は０．１質量％以上０．４質量％未満が好ましい。尚、飼料中のコール酸又はその塩の含有量が多い（例えば、０．５質量％超、中でも、０．５質量％超１質量％）ほど肝障害（肝線維化等）を誘導しやすい場合もある。 The content of cholic acid or its salt in feed is not particularly limited as long as the effects of the present invention are not impaired, but it is usually 0.01 to 2% by mass, preferably 0.1 to 1% by mass, Preferably, it is less than 0.5% by mass (for example, 0.1 to 0.45% by mass), and particularly preferably 0.2 to 0.4% by mass.
It is preferable that the content of cholic acid or its salt in feed is low (eg, more than 0% by mass and less than 2% by mass). The lower the amount of cholic acid, the better the NAFLD model mouse is obtained. Specifically, for example, as the content of cholic acid or a salt thereof in the feed is smaller (for example, less than 0.5% by mass or less than 0.4% by mass), the mouse receiving the feed has a weight or an epididymal fat Etc. increase weight / weight of visceral fat or show insulin resistance. Specifically, the content of cholic acid or a salt thereof in feed is, for example, preferably 0.001% by mass or more and less than 0.5% by mass or 0.1% by mass or more and less than 0.4% by mass. In addition, as the content of cholic acid or its salt in feed (for example, more than 0.5% by mass, in particular, more than 0.5% by mass and 1% by mass) tends to induce liver damage (hepatic fibrosis etc.) In some cases.
　コレステロール
　コレステロールは、公知の化学合成、微生物培養、酵素反応等により製造することもでき、また市販品を購入して入手することもできる。市販品としては、例えば、クローダジャパン株式会社製のＣＯＬＥＳＴＥＲＯＬ　ＮＦ（商品名）、日本精化株式会社製のコレステロールＪＳＱＩ（商品名、日本薬局方コレステロール）、日本水産株式会社製のニッスイマリンコレステロール（商品名）等が挙げられる。飼料中のコレステロール含有量は、本発明の効果を妨げない限り特に限定されないが、通常０．０１～５質量％であり、好ましくは０．１～３質量％であり、特に好ましくは１～１．５質量％である。飼料中のコレステロール含有量は高い（例えば、１質量％以上）ことが好ましい。飼料中のコレステロール含有量が高いほど、より優れたＮＡＦＬＤモデルマウスが得られる。具体的には、例えば、飼料中のコレステロール含有量が高いほど、飼料を摂取したマウスは、高い血中ＡＬＴ値を示し、又は高度の肝線維化を示す。飼料中のコレステロール含有量は、具体的には、例えば０．０１～２．５質量％が好ましい。 Cholesterol Cholesterol can also be produced by known chemical synthesis, microorganism culture, enzyme reaction and the like, and can also be obtained by purchasing commercially available products. Examples of commercially available products include COLESTEROL NF (trade name) manufactured by Croda Japan Co., Ltd., cholesterol JSQI (trade name Japan Pharmacopoeia cholesterol) manufactured by Nippon Seika Co., Ltd., Nissui Marine Cholesterol manufactured by Japan Fisheries Co., Ltd. (Product Name) etc. The cholesterol content in feed is not particularly limited as long as the effects of the present invention are not impaired, but it is usually 0.01 to 5% by mass, preferably 0.1 to 3% by mass, particularly preferably 1 to 1 .5 mass%. The cholesterol content in the feed is preferably high (eg, 1% by mass or more). The higher the cholesterol content in the feed, the better the NAFLD model mice obtained. Specifically, for example, the higher the cholesterol content in the feed, the higher the feed intake, the higher the blood ALT value or the higher the degree of hepatic fibrosis. Specifically, the content of cholesterol in feed is, for example, preferably 0.01 to 2.5% by mass.
　標準飼料
　標準飼料は、マウスの飼育に使用され得るものであればどのようなものでもよいが、通常、一般成分（例えば、水分、粗タンパク質、粗脂質、粗灰分、粗繊維、可溶性無窒素物又はこれらの２種以上（好ましくは全部））、ビタミン類（ビタミンＡ、ビタミンＤ３、ビタミンＥ、ビタミンＫ３、ビタミンＢ１、ビタミンＢ２、ビタミンＣ、ビタミンＢ６、イノシトール、ビオチン、パントテン酸、ナイアシン、コリン、葉酸、又はこれらの２種以上（好ましくは全部）の組合せ等）、ミネラル類（カルシウム、リン、マグネシウム、ナトリウム、カリウム、鉄、アルミニウム、銅、亜鉛、コバルト、マンガン、又はこれらの２種以上（好ましくは全部）の組合せ等）、アミノ酸（イソロイシン、ロイシン、リジン、メチオニン、シスチン、フェニルアラニン、チロシン、ステオニン、トリプトファン、バリン、アルギニン、ヒスチジン、アラニン、アスパラギン酸、グルタミン酸、グリシン、プロリン、セリン、又はこれらの２種以上（好ましくは全部）の組合せ等）を含有する。
　標準飼料として、オリエンタル酵母工業株式会社製のＭＦ、ＭＮＦ、ＣＭＦ、ＣＲ－ＬＰＦ、若しくはＣＲＦ－１等、又は日本クレア株式会社製のＣＥ－２等が挙げられるが、中でもＭＦが好ましい。
　標準飼料は、当技術分野で通常用いられている実験動物用飼料と言い換えることができる。 Standard Feed Standard Feed may be anything as long as it can be used for rearing mice, but it is usually a common ingredient (eg, water, crude protein, crude lipid, crude ash, crude fiber, soluble nitrogen-free) Or two or more of them (preferably all), vitamins (vitamin A, vitamin D3, vitamin E, vitamin K3, vitamin B1, vitamin B2, vitamin C, vitamin B6, inositol, biotin, pantothenic acid, niacin, choline , Folic acid, or a combination of two or more (preferably all) of them, minerals (calcium, phosphorus, magnesium, sodium, potassium, iron, aluminum, copper, zinc, cobalt, manganese, or two or more of them) (Preferably all combinations etc.), amino acids (isoleucine, leucine, lysine, methionine, cis) Emissions, containing phenylalanine, tyrosine, Suteonin, tryptophan, valine, arginine, histidine, alanine, aspartic acid, glutamic acid, glycine, proline, serine, or two or more of these combinations of (preferably all)).
Examples of standard feeds include MF, MNF, CMF, CR-LPF, CRF-1 and the like manufactured by Oriental Yeast Co., Ltd., and CE-2 and the like manufactured by CLEA Japan, Inc. Among them, MF is preferable.
Standard diets can be reworded as laboratory animal diets that are commonly used in the art.
　飼料中の標準飼料含有量は、本発明の効果を妨げない限り特に限定されないが、通常５０～９５質量％であり、好ましくは５５～９０質量％であり、特に好ましくは６０～８５質量％である。 The standard feed content in the feed is not particularly limited as long as the effects of the present invention are not impaired, but it is usually 50 to 95% by mass, preferably 55 to 90% by mass, and particularly preferably 60 to 85% by mass. is there.
　脂質エネルギー比率
　脂質エネルギー比率は、次の式で求めることができる。
　脂質エネルギー比率（％）＝{脂質量（グラム）×９キロカロリー／総エネルギー（キロカロリー）}×１００
　飼料の脂質エネルギー比率は高い（例えば、３０％以上）ことが好ましい。飼料の脂質エネルギー比率が高いほど、より優れたＮＡＦＬＤモデルマウスが得られる。具体的には、例えば、飼料の脂質エネルギー比率は高いほど、飼料を摂取したマウスは、体重が増加し、又は高度の肝線維化を示す。飼料の脂質エネルギー比率は具体的には、例えば３０～５０％が好ましい。それ以上であってもよいが、より大きな効果を得ることが困難である。
　飼料の脂質エネルギー比率を増やすために、例えば、ヤシ油、パーム油、大豆油、綿実油、落花生油、米油等の植物油若しくはその油かす；ショートニング；マーガリン；硬化油；又は、動物性油等の油脂を飼料に含めてもよい。油脂は、その構成脂肪酸として、トランス脂肪酸、又は飽和脂肪酸を含有する油脂であることが好ましい。例えば、市販されている油脂を購入することで、油脂を入手できる。これらの油脂を含む飼料をマウスに与えることにより、優れたＮＡＦＬＤモデルマウスが得られ得る。具体的には、これらの油脂を含む飼料をマウスに与えることにより、例えば、高度な線維化を示す又は体重若しくは体重当たりの内臓脂肪の重さが増加しているＮＡＦＬＤモデルマウスが短期間で得られる。
　飼料中の油脂含有量は、本発明の効果を妨げない限り特に限定されないが、通常１５～６５質量％であり、好ましくは２０～６０質量％であり、特に好ましくは２５～５０質量％である。 Lipid energy ratio The lipid energy ratio can be determined by the following equation.
Lipid energy ratio (%) = {lipid amount (gram) x 9 kcal / total energy (kcal)} x 100
Preferably, the lipid energy ratio of the feed is high (eg, 30% or more). The higher the lipid energy ratio of the feed, the better the NAFLD model mice obtained. Specifically, for example, the higher the lipid energy ratio of the feed, the mouse receiving the feed gains weight or exhibits a high degree of liver fibrosis. Specifically, the lipid energy ratio of feed is, for example, preferably 30 to 50%. Although it may be more than that, it is difficult to obtain a larger effect.
Vegetable oil such as coconut oil, palm oil, soybean oil, cottonseed oil, peanut oil, rice oil etc. or shortening thereof; shortening; margarine; hardened oil; or animal oil etc. to increase the lipid energy ratio of feed Fats and oils may be included in the feed. The fats and oils are preferably fats and oils containing trans fatty acids or saturated fatty acids as constituent fatty acids. For example, fats and oils can be obtained by purchasing commercially available fats and oils. By feeding the diet containing these fats and oils to mice, excellent NAFLD model mice can be obtained. Specifically, feeding a feed containing these fats and oils to mice allows, for example, a NAFLD model mouse showing a high degree of fibrosis or increasing the weight of visceral fat per body weight or body weight to be obtained in a short period of time. Be
The content of fat and oil in feed is not particularly limited as long as the effects of the present invention are not impaired, but is usually 15 to 65% by mass, preferably 20 to 60% by mass, and particularly preferably 25 to 50% by mass. .
　また、本発明の飼料は、必要に応じて、一般飼料、精製飼料、滅菌飼料、特殊配合飼料等の上記標準飼料以外の実験動物用飼料；保存剤、抗酸化剤、安定化剤、酸化防止剤溶剤、溶解補助剤、懸濁化剤、等張化剤等の飼料の分野において通常用いられる任意の公知の添加剤；及び／又は薬理学的に許容される添加剤を必要に応じて用いることもできる。これらを、目的とする飼料形態に応じて、単独で又は２種以上を組み合わせて使用することができる。これらは、市販品を使用することができる。 In addition, the feed of the present invention may be, if necessary, a feed for laboratory animals other than the above standard feed such as general feed, purified feed, sterile feed, specially formulated feed, etc .; preservative, antioxidant, stabilizer, antioxidant Optionally using any known additive commonly used in the field of feed such as pharmaceutical solvents, solubilizers, suspending agents, tonicity agents, etc .; and / or pharmacologically acceptable additives as required It can also be done. These can be used alone or in combination of two or more depending on the intended feed form. A commercial item can be used for these.
　保存剤としては、特に限定されないが、例えば、パラオキシ安息香酸エチル、クロロブタノール、ベンジルアルコール、デヒドロ酢酸ナトリウム、ソルビン酸等が挙げられる。抗酸化剤としては、特に限定されないが、例えば、亜硫酸ナトリウム、アスコルビン酸等が挙げられる。安定化剤としては、特に限定されないが、例えば、カゼイン、カゼインナトリウム塩等が挙げられる。酸化防止剤としては、例えばｔ－ブチルヒドロキノン、ブチルヒドロキシアニソール、ブチルヒドロキシトルエン、及びα－トコフェロール、並びにそれらの誘導体が挙げられる。 The preservative is not particularly limited, and examples thereof include ethyl parahydroxybenzoate, chlorobutanol, benzyl alcohol, sodium dehydroacetate, sorbic acid and the like. The antioxidant is not particularly limited, and examples thereof include sodium sulfite, ascorbic acid and the like. The stabilizer is not particularly limited, and examples thereof include casein, casein sodium salt and the like. Antioxidants include, for example, t-butylhydroquinone, butylhydroxyanisole, butylhydroxytoluene, and α-tocopherol, and derivatives thereof.
　飼料は固形状、液体、乳化剤、ペースト、ゲル、粉末、錠剤、顆粒、ペレット、カプセル、シロップ、懸濁液又はスティック状に加工されていてもよい。固体状である場合は、略円柱状であってもよい。これらは公知方法で製造されてよい。 The feed may be processed into solid, liquid, emulsifier, paste, gel, powder, tablets, granules, pellets, capsules, syrups, suspensions or sticks. When it is solid, it may be substantially cylindrical. These may be manufactured by known methods.
　本発明の飼料は、肝臓中のトリグリセリド蓄積作用、血中のＡＬＴ値を増加させる作用等を有する。また、飼料中にコレステロール等脂質を少量含むことで、飼料を摂取したマウスの体重を増加させることができる。
　本発明の飼料は、これを摂取したマウスにＮＡＦＬＤの病態（特に、肝線維化）を引き起こすことができる。 The feed of the present invention has a triglyceride accumulation action in the liver, an action to increase the ALT level in blood, and the like. In addition, by containing a small amount of lipids such as cholesterol in the feed, it is possible to increase the body weight of the mouse that has received the feed.
The feed of the present invention can cause the pathological condition of NAFLD (in particular, liver fibrosis) in mice receiving it.
〔飼料の製造方法〕
　本発明の飼料は、上記含有成分を、適量添加し、公知又は自体公知の方法に従って、混練、撹拌及び／又は混合することにより製造することができる。 [Method of producing feed]
The feed of the present invention can be produced by adding an appropriate amount of the above-mentioned components and kneading, stirring and / or mixing according to a known method or a method known per se.
２．ＮＡＦＬＤモデル動物の作製方法
　本発明は、マウスに本発明の飼料を与えて飼育する工程を含むことを特徴とする非アルコ－ル性脂肪肝疾患モデルマウスの作製方法を提供する。 2. Method for Producing NAFLD Model Animal The present invention provides a method for producing a non-alcoholic fatty liver disease mouse model, which comprises the step of feeding the feed of the present invention to a mouse for breeding.
　マウス
　マウスは、遺伝子改変マウスでないことが好ましい。マウスの種類としては、例えば、Ａ／Ｊ、Ｃ５７ＢＬ／６Ｊ、ＴＳＮＯ（Ｔｓｕｍｕｒａ－Ｓｕｚｕｋｉ　ｎｏｎ－ｏｂｅｓｅ）、 ＴＳＯＤ（Ｔｓｕｍｕｒａ－Ｓｕｚｕｋｉ　ｏｂｅｓｅ　ｄｉａｂｅｔｅｓ）Ｃ５７ＢＬ／６Ｎ、Ｃ３Ｈ／ＨｅＮ、Ｃ３Ｈ／ＨｅＪ、ＢＡＬＢ／ｃ、ＦＶＢ／Ｎ、１２９＋Ｔｅｒ／Ｓｖ、ＮＯＤ／Ｓｈｉ、ＮＣ、ＦＧＳ／Ｎｇａ、ＣＢＡ／ＪＮ、ＤＢＡ／１、ＤＢＡ／２、ＮＣ／Ｎｇａ、ＡＫＲ／Ｊ等の近交系又はＩＣＲ、ｄｄＹ等のクローズドコロニーが挙げられ、中でもＡ／Ｊ、又はＴＳＮＯが好ましい。
　例えば、Ｃ５７ＢＬ／６Ｊは標準モデルとして、Ａ／Ｊはアジア人モデル又は痩せ型モデルとして、ＴＳＮＯは強線維化モデルとして好適である。Ａ／Ｊに関して、より詳細には、アジア人は肥満型でなくてもＮＡＦＬＤになると報告されているため、肥満抵抗性を示すＡ／Ｊは、アジア人モデル又は痩せ型モデルとして好適である。ＴＳＮＯに関して、より詳細には、食餌のみで誘導できる肝線維化モデルとして好適であることが後述の試験例４によって裏付けられている。 The mouse is preferably not a genetically modified mouse. As the type of mouse, for example, A / J, C57BL / 6J, TSNO (Tsumura-Suzuki non-obese), TSOD (Tsumura-Suzuki obese diabetes) C57BL / 6N, C3H / HeN, C3H / HeJ, BALB / c, Inbred lines such as FVB / N, 129 + Ter / Sv, NOD / Shi, NC, FGS / Nga, CBA / JN, DBA / 1, DBA / 2, NC / Nga, AKR / J, etc. or closed such as ICR, ddY A colony is mentioned and A / J or TSNO is preferable among them.
For example, C57BL / 6J is suitable as a standard model, A / J as an Asian model or lean model, and TSNO as a strong fibrosis model. As for A / J, more specifically, Asians are reported to become NAFLD even if they are not obese, so A / J showing obesity resistance is suitable as an Asian model or lean model. With regard to TSNO, more particularly, it is supported by Test Example 4 described below that it is suitable as a diet-induced hepatic fibrosis model.
　摂餌量
　通常、飼料をマウスケージ内又はケージのかぶせ蓋（網状）に、常法に従って、適量乗せることで、マウスは飼料を自由摂取する。１日当たりのマウスの摂餌量は、通常約３～５グラムであるが、これに限定されず、マウス個体が飼育条件下で実際に食べることができる量であってよい。 Feeding amount Usually, the mouse freely feeds the feed by placing the feed in a mouse cage or in a cage lid (reticular) according to a conventional method. The daily food intake of a mouse is usually about 3 to 5 grams, but is not limited thereto, and may be an amount that a mouse individual can actually eat under breeding conditions.
　飼料を与える期間
　飼料を与え始める時期は、離乳後であれば特に限定されないが、例えば４週齢以上であることが好ましく、５～１０週齢であることがより好ましい。飼料を与える期間は、例えば６週間以上であることが好ましく、９週間以上であることがより好ましく、１０週間以上であることがより好ましく、１１週間以上であることがより好ましい。飼料を与える期間が長いほど（例えば９週間以上）、より優れたＮＡＦＬＤモデルマウスが得られる。具体的には、例えば、飼料を与える期間が長いほど、飼料を摂取したマウスは高度の肝線維化を示す。飼料を与える期間は、好ましくは６～５２週間であり、より好ましくは１０～２６週間又は２６～５２週間である。飼料を与える期間は、コール酸量が少ないほど長期間であることが好ましい。飼料を与える期間は、コレステロールが少ないほど長期間であることが好ましい。飼料を与える期間は、脂質エネルギー比率が低いほど長期間であることが好ましい。
　マウスの飼育環境は、温度２０～２５℃、湿度５０～６０％、１２時間は明期、１２時間は暗期サイクルに設定された室内であることが好ましい。 Feeding period is not particularly limited as long as feeding is started after weaning, but for example, it is preferably 4 weeks or more, and more preferably 5-10 weeks. The feed period is, for example, preferably 6 weeks or more, more preferably 9 weeks or more, more preferably 10 weeks or more, and still more preferably 11 weeks or more. The longer the feeding period (eg, 9 weeks or more), the better the NAFLD model mouse is obtained. Specifically, for example, the longer the feed is given, the more the mice receiving the feed, the higher the degree of liver fibrosis. The feed period is preferably 6 to 52 weeks, more preferably 10 to 26 weeks or 26 to 52 weeks. The feed period is preferably as long as the amount of cholic acid is small. The lower the cholesterol, the longer the feed period. It is preferable that the feeding period is longer as the lipid energy ratio is lower.
The breeding environment of the mouse is preferably a room with a temperature of 20 to 25 ° C., a humidity of 50 to 60%, a 12 hour light period, and a 12 hour dark cycle.
３．ＮＡＦＬＤモデル動物
　非アルコール性脂肪肝疾患（ＮＡＦＬＤ）は、病理組織学的に大滴性の肝脂肪変性を基本として、肝細胞障害（風船様変化）や肝線維化がみられる非アルコール性脂肪肝炎（ＮＡＳＨ）と、病態の進行がみられない非アルコール性脂肪肝（ＮＡＦＬ）に大別される。
　本発明のＮＡＦＬＤモデルマウスは、遺伝子改変技術を使用しないで作製され、よりヒトに近く、利便性が高いＮＡＦＬＤモデル動物である点において、特に優れている。
　マウスは、他の実験動物（例えば、ラットなど）と比較し、異なる性質を有する。例えば、マウスは胆嚢を有する点でヒトに近い。また、マウスは、例えば個体の大きさが小さく、餌の量が少なくてすむ、取り扱いやすい等の点から利便性が高い。
　本発明のＮＡＦＬＤモデルマウスがＮＡＦＬＤ病態を示すことは、例えば、公知又は自体公知の生化学的測定手法により、血中トリグリセリド値、肝臓トリグリセリド値、血中コレステロール値、血中グルコース値、血中ＡＬＴ値、血中インスリン値、又は血中レプチン値等を測定し、ヒトのＮＡＦＬＤ病態と同じような挙動を示すか否かで簡単に確認することができる。同じような挙動とは、例えば、無疾患群（本発明の飼料を投与していない群）の血中トリグリセリド値、肝臓トリグリセリド値、血中コレステロール値、肝臓コレステロール値、血中グルコース値、血中ＡＬＴ値、血中インスリン値、インスリン抵抗性指標、又は血中レプチン値等と比較したこれら値の傾向（減少、又は増加）が同じであること等という。
　例えば、血中インスリン値、血中グルコース値、血中コレステロール値、血中ＡＬＴ値及び肝臓トリグリセリド値のいずれか１以上について、本発明の飼料を投与していない群と比較して有意に増加している場合、本発明の飼料を投与した群は、ＮＡＦＬＤモデルマウスとして有用である。
　また、例えば、体重又は体重当たりの内臓脂肪の重さの増加幅について、本発明の飼料を投与していない群と比較して有意に少なくない場合、本発明の飼料を投与した群は、ＮＡＦＬＤモデルマウスとして有用であり、本発明の飼料を投与していない群と比較して大きい場合、本発明の飼料を投与した群は、ＮＡＦＬＤモデルマウスとしてより優れている。
　本発明のＮＡＦＬＤモデルマウスがＮＡＳＨ病態を示すことは、例えば、ヘマトキシリン・エオジン（ｈｅｍａｔｏｘｙｌｉｎ　ａｎｄ　ｅｏｓｉｎ，ＨＥ）染色、又はアザン（Ａｚａｎ）染色等により肝組織を染色し、「ＮＡＳＨ　Ｃｌｉｎｉｃａｌ　Ｒｅｓｅａｒｃｈ　Ｎｅｔｗｏｒｋ　Ｓｃｏｒｉｎｇ　Ｓｙｓｔｅｍ」の診断基準に従い病理観察することにより確認することができる。具体的には、肝組織を病理観察して、下記表１に示すＮＡＦＬＤ　ａｃｔｉｖｉｔｙ　ｓｃｏｒｅ（ＮＡＳ）の合計点数８点中、０～２点はＮＡＳＨではない、３～４点は判定保留、５点以上はＮＡＳＨと判断することができる。 3. NAFLD model animal Non-alcoholic fatty liver disease (NAFLD) is a non-alcoholic steatohepatitis with hepatocellular injury (balloon-like change) and hepatic fibrosis, based on histopathologically large hepatic steatosis. (NASH) and non-alcoholic fatty liver (NAFL), in which progression of pathological condition is not observed.
The NAFLD model mouse of the present invention is particularly excellent in that it is a human-friendly, highly convenient NAFLD model animal that is produced without using genetic modification technology.
Mice have different properties compared to other experimental animals (eg, rats etc.). For example, mice are closer to humans in having a gallbladder. In addition, the mouse is highly convenient, for example, in that the size of the individual is small, the amount of food can be small, and it is easy to handle.
It is known that the NAFLD model mouse of the present invention shows the pathological condition of NAFLD, for example, blood triglyceride level, liver triglyceride level, blood cholesterol level, blood glucose level, blood ALT level by a known biochemical method per se. The level, insulin level in blood, leptin level in blood, etc. can be measured, and it can be easily confirmed whether or not the behavior similar to that of human NAFLD is exhibited. Similar behavior is, for example, blood triglyceride level, liver triglyceride level, blood cholesterol level, liver cholesterol level, blood glucose level, blood glucose level in the non-diseased group (group not receiving the feed of the present invention) It is said that the tendency (decrease or increase) of these values in comparison with ALT value, blood insulin value, insulin resistance index, blood leptin value etc. is the same.
For example, any one or more of blood insulin level, blood glucose level, blood cholesterol level, blood ALT level and liver triglyceride level is significantly increased as compared to the group not receiving the feed of the present invention. When the group is administered, the group to which the feed of the present invention has been administered is useful as a NAFLD model mouse.
Also, for example, when the increase in the weight of visceral fat per body weight or body weight is not significantly less than the group not receiving the feed of the present invention, the group receiving the feed of the present invention is NAFLD When it is useful as a model mouse and large compared to the group not receiving the feed of the present invention, the group receiving the feed of the present invention is superior as a NAFLD model mouse.
The fact that the NAFLD model mouse of the present invention shows NASH disease states that liver tissue is stained by, for example, hematoxylin and eosin (HE) staining or azan (Azan) staining, and the "NASH Clinical Research Network Scoring System". This can be confirmed by pathological observation according to the diagnostic criteria of Specifically, the liver tissue is pathologically observed, and 0 to 2 points are not NASH, 3 to 4 points are judged pending, 5 points out of 8 points in the total points of NAFLD activity score (NAS) shown in Table 1 below. The above can be judged as NASH.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　本発明のＮＡＦＬＤモデルマウスは、より好ましくはＮＡＳＨ病態の１つとしての肝の線維化を示し得る。中でもマウスとしてＴＳＮＯマウスを採用することは、肝の線維化が示され得る点で好ましい。肝の線維化の評価は、例えば、肝組織を病理観察して、下記表２に示す判断基準に従って行い得る。 The NAFLD model mouse of the present invention may more preferably exhibit hepatic fibrosis as one of NASH pathological conditions. Above all, adopting TSNO mice as mice is preferable in that fibrosis of the liver can be shown. Assessment of liver fibrosis can be performed, for example, according to the criteria shown in Table 2 below, by pathologically observing liver tissue.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　本発明のＮＡＦＬＤモデルマウスがＮＡＦＬ病態を示すことは、例えば、組織診断で脂肪肝を認め、かつ肝細胞障害（風船様変性）や線維化を認めないことにより確認することができる。 The fact that the NAFLD model mouse of the present invention exhibits the NAFL pathological condition can be confirmed, for example, by recognizing fatty liver by tissue diagnosis and not recognizing hepatocellular injury (balloon-like degeneration) or fibrosis.
　本発明は、本発明の効果を奏する限り、本発明の技術的範囲内において、上記の構成を種々組み合わせた態様を含む。 The present invention includes embodiments in which the above-described configurations are variously combined within the technical scope of the present invention as long as the effects of the present invention can be obtained.
　次に、実施例を挙げて本発明をさらに具体的に説明するが、本発明はこれらの実施例により何ら限定されるものではなく、多くの変形が本発明の技術的思想内で当分野において通常の知識を有する者により可能である。 EXAMPLES The present invention will next be described in more detail by way of examples, which should not be construed as limiting the present invention in any way, and many modifications may be made within the technical scope of the present invention. It is possible by the person of ordinary knowledge.
１．試験例１
（１）動物モデル及び実験デザイン
　８週齢雄性Ｃ５７ＢＬ／６Ｊマウス（Ｂ６マウス）１５匹及び８週齢雄性Ａ／Ｊマウス１５匹（いずれも日本ＳＬＣ、静岡）を購入し、初めの１週間は標準飼料（ＭＦ；オリエンタル酵母、東京）を摂取させ飼育環境に順化させた。Ｂ６マウス及びＡ／Ｊマウスは順化後の９週齢時から、標準飼料であるＭＦを摂取させたＮｏｒｍａｌ群（Ｎ群）、ＭＦに粉末パーム油２８．７５質量％、コレステロール１．２５質量％及びコール酸ナトリウム塩０．５質量％を添加した高脂肪飼料を摂取させた低コレステロール群（ＬＣ群）、ＭＦにパーム油２７．５質量％、コレステロール２．５質量％及びコール酸ナトリウム塩０．５質量％を添加した高脂肪飼料を摂取させた高コレステロール群（ＨＣ群）の３群各４～６匹ずつに分け、１８週齢まで飼育した。飼料はオリエンタル酵母に作製を依頼し、購入した。それぞれの飼料組成及びエネルギーは下記表３に示す。食餌及び水は自由摂取とし、飼育期間中、体重測定を１週間に２回、摂食量測定を２日に１回行った。
　すべての動物は、温度２４℃、湿度５５％、７～１９時は明期、１９～７時は暗期サイクルに設定された室内で飼育した。なお、本動物実験は「奈良女子大学動物実験に関する指針」ならびに「実験動物の飼育及び保管等に関する基準（昭和５５年３月総理府告示第６号）」に則して実施した。 1. Test Example 1
(1) Animal model and experimental design 15 8-week-old male C57BL / 6J mice (B6 mice) and 15 8-week-old male A / J mice (all from Japan SLC, Shizuoka) were purchased, and the first week was Standard feed (MF; Oriental Yeast, Tokyo) was ingested to acclimate to the breeding environment. B6 mice and A / J mice are normal group (group N) fed MF as a standard diet from 9 weeks of age after acclimatization, 28.75% by mass of powdered palm oil in MF, 1.25 mass of cholesterol % And low cholesterol group (LC group) fed a high-fat diet supplemented with 0.5% by weight of sodium cholate, MF 27.5% by weight of palm oil, 2.5% by weight of cholesterol and cholate sodium salt The high-cholesterol group (HC group) which received a high-fat diet to which 0.5% by mass had been added was divided into 4 to 6 animals each for 3 groups and reared until 18 weeks of age. The feed was purchased from Oriental yeast for preparation. The respective feed composition and energy are shown in Table 3 below. Food and water were free intake, and during the breeding period, body weight measurement was performed twice a week and food intake measurement was performed once every two days.
All animals were kept in a room set at a temperature of 24 ° C., a humidity of 55%, a light period of 7-19 o'clock and a dark cycle of 19-7 o'clock. The present animal experiment was conducted in accordance with "Guideline on animal experiment of Nara Women's University" and "Standard for breeding and storage of experimental animal (announcement No. 6 of the Prime Minister's notice in March 1959)".
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
（２）動物処理
　Ｂ６マウス及びＡ／Ｊマウスは１８週齢時の屠殺前に８時間絶食させたのち、ソムノペンチル（商品名、共立製薬株式会社、一般名はペントバルビタールナトリウム）麻酔下で開腹処置を行い、シリンジを用いて腹部大静脈又は心臓から採血した。直ちに肝臓を摘出し、０．９％(ｗ／ｖ)食塩水で洗浄後重量を測定した。肝臓の一部を切除し、１０％(ｗ／ｖ)中性ホルマリン液で固定した。また、肝臓の一部（０．５ｇ）を凍結保存して、後述するように脂質の抽出を行った。 (2) Animal treatment B6 mice and A / J mice are fasted for 8 hours before slaughtering at 18 weeks of age and then open treated under anesthesia with Somnopentyl (trade name, Kyoritsu Pharmaceutical Co., Ltd., general name: pentobarbital sodium) And blood was drawn from the abdominal vena cava or heart using a syringe. The liver was immediately removed, washed with 0.9% (w / v) saline and weighed. A part of the liver was excised and fixed with 10% (w / v) neutral formalin solution. In addition, a portion (0.5 g) of the liver was cryopreserved and lipid extraction was performed as described later.
（３）血清・肝臓生化学的測定
　卓上遠心機（テーブルトップマイクロ冷却遠心機３５００、久保田商事、東京）で４℃、３０００ｒｐｍで１０～１５分間、上記採取した血液を遠心分離し、上清（血清）を凍結保存した。血清中の、総コレステロール、ALTを測定した。各項目の吸光度の測定にはライフサイエンス用紫外可視分光光度計　Ｖ－６３０ＢＩＯ（日本分光株式会社 ＪＡＳＣＯ　Ｃｏｒｐｏｒａｔｉｏｎ、東京）を使用した。各項目の測定原理及び方法を以下に示す。 (3) Serum and Liver Biochemical Measurement The collected blood is centrifuged at 3000 rpm for 10 to 15 minutes at 4 ° C. with a table-top centrifuge (tabletop micro refrigerated centrifuge 3500, Kubota, Tokyo) for 10 minutes. Serum was stored frozen. Total cholesterol and ALT in serum were measured. An ultraviolet-visible spectrophotometer V-630BIO (JASCO Corporation, JASCO Corporation, Tokyo) for life sciences was used to measure the absorbance of each item. The measurement principle and method of each item are shown below.
〔血清中の総コレステロール〕
　コレステロールオキシターゼ・ＤＡＯＳ法を用いた測定キットであるコレステロールＥ－テストワコー（和光純薬工業）を使用し、説明書通りの方法で血清中の総コレステロールの測定を行った。
　測定は１検体につき２回行い、その平均値を測定した。 [Total cholesterol in serum]
Measurement of total cholesterol in serum was carried out according to the method described in the instruction using Cholesterol E-Test Wako (Wako Pure Chemical Industries), which is a measurement kit using the cholesterol oxidase / DAOS method.
The measurement was performed twice for one sample, and the average value was measured.
〔血清中のＡＬＴ〕
　（ＰＯＰ・ＴＯＯＳ）法を用いた測定キットであるトランスアミナーゼＣ－ＩＩテストワコー（和光純薬工業）を使用して、説明書通りの方法で血清ＡＬＴ濃度の測定を行った。
　測定は１検体につき２回行い、その平均値を測定値とした。 [ALT in serum]
Using the transaminase C-II Test Wako (Wako Pure Chemical Industries, Ltd.), which is a measurement kit using the (POP · TOOS) method, the serum ALT concentration was measured according to the method described in the instruction.
The measurement was performed twice for one sample, and the average value was taken as the measurement value.
〔肝臓トリグリセリド〕
　凍結保存した肝臓０．５ｇを用いて肝臓脂質を抽出し、ＴＧ濃度を測定した。抽出はＦｏｌｃｈらの方法により行った。
　肝組織０．５ｇをホモジナイズし、メタノール・クロロホルム混合液（容量比１：２）約５０ｍＬでメスフラスコに洗いながら移し入れ、４０℃で３０分間振盪反応後、室温に戻してからメタノール・クロロホルム混合液を加えて５０ｍＬとした。濾過後、蒸留水約９ｍＬを加えて静かに混合し水層は捨てた。 [Liver triglyceride]
Liver lipids were extracted using 0.5 g of cryopreserved liver, and the TG concentration was measured. Extraction was performed by the method of Folch et al.
0.5 g of liver tissue is homogenized, transferred while transferring to a measuring flask with a methanol / chloroform mixed solution (volume ratio 1: 2) with about 50 mL, shaken at 40 ° C. for 30 minutes, returned to room temperature and mixed with methanol / chloroform The solution was added to 50 mL. After filtration, about 9 mL of distilled water was added and mixed gently, and the aqueous layer was discarded.
＜測定方法＞
　室温に戻した後、サンプルを試験管に取り、ヘキサンで希釈したサンプルからさらにサンプルを試験管に取り窒素乾固後、イソプロパノール１００μＬを加えてよく混合した。これを測定に用いた。測定にはトリグリセリドＥ－テストワコー（和光純薬工業）を用いた。 <Measurement method>
After returning to room temperature, the sample was taken in a test tube, and from the sample diluted with hexane, another sample was taken in a test tube and dried in nitrogen, and then 100 μL of isopropanol was added and mixed well. This was used for the measurement. Triglyceride E-Test Wako (Wako Pure Chemical Industries) was used for the measurement.
（４）病理組織学検討
　病理組織学的所見は群分けを伏せて行うブラインドにより、すべて病理医が評価した。 (4) Pathohistological examination Pathological findings were all evaluated by a pathologist by the blind performed by grouping.
〔ヘマトキシリン・エオジン（ｈｅｍａｔｏｘｙｌｉｎ　ａｎｄ　ｅｏｓｉｎ，ＨＥ）染色〕
　肝組織が包理されたパラフィンブロックをミクロソームで４μｍの厚さに薄切りし、脱パラフィン処理し水洗した後、マイヤーのヘマトキシリン液にて５分間染色した。水洗後、エオジン液にて２～３分染色し、アルコールで分別した後、脱水・透徹・封入した。 [Hematoxylin and eosin (HE) staining]
The paraffin block in which the liver tissue was embedded was sliced with a microsome to a thickness of 4 μm, deparaffinized and washed with water, and then stained for 5 minutes with Mayer's hematoxylin solution. After washing with water, it was stained with eosin solution for 2 to 3 minutes, fractionated with alcohol, and then dehydrated, cleared and sealed.
〔アザン（Ａｚａｎ）染色〕
　肝臓組織が包理されたパラフィンブロックをミクロトームで４μｍの厚さに薄切し、脱パラフィン処理し水洗した後、１０％(ｗ／ｖ)重クロム酸カリウム水溶液と１０％(ｗ／ｖ)トリクロール酢酸水溶液を等量混合した媒染剤に１０～２０分浸した。再び水洗した後、アゾカルミンＧ液に室温で３０分以上浸した。蒸留水で水洗し、アニリン・アルコールで数秒間分別し、酢酸・アルコールで分別を停止し、水洗後、鏡検して染色状態を確認した。その後、５％(ｗ／ｖ)リンタングステン酸水溶液に１時間～１晩浸した。水洗後、アニリン青・オレンジＧ混合液に３０～６０分浸し、１００質量％アルコールで分別脱水し、３槽以上のキシロールで透徹、封入した。 [Azan (Azan) staining]
A paraffin block embedded with liver tissue is sliced with a microtome to a thickness of 4 μm, deparaffinized and washed with water, and then 10% (w / v) aqueous potassium dichromate solution and 10% (w / v) trily It was immersed for 10 to 20 minutes in a mordant mixed with an equal amount of aqueous solution of chloroacetic acid. After washing again with water, it was immersed in azocarmine G solution at room temperature for 30 minutes or more. It was washed with distilled water, separated with aniline and alcohol for several seconds, stopped with acetic acid and alcohol, washed with water, and microscopically checked to confirm the dyed state. Then, it was immersed in a 5% (w / v) aqueous solution of phosphotungstic acid for 1 hour to 1 night. After washing with water, it was immersed in a mixed solution of aniline blue and orange G for 30 to 60 minutes, fractionated and dehydrated with 100% by mass alcohol, and cleared and sealed with three or more tanks of xylol.
〔肝病変の評価〕
　群分けを伏せて行うブラインドにより、すべて病理医が評価した。診断基準はＫｌｅｉｎｅｒらが提唱した「ＮＡＳＨ　Ｃｌｉｎｉｃａｌ　Ｒｅｓｅａｒｃｈ　Ｎｅｔｗｏｒｋ　Ｓｃｏｒｉｎｇ　Ｓｙｓｔｅｍ」の診断基準に従った。これはｓｔｅａｔｏｓｉｓ（脂肪沈着）、ｌｏｂｕｌａｒ　ｉｎｆｌａｍｍａｔｉｏｎ（小葉内炎症）、ｈｅｐａｔｏｃｉｔｅｓ　ｂａｌｌｏｏｎｉｎｇ（肝細胞の風船様変化）を上記表１に示す分類に従ってスコア化し、ＮＡＳＨの程度を評価する方法である。さらに、脂肪沈着、小葉内炎症、肝細胞の風船様変化の３カテゴリーを合わせてＮＡＦＬＤ　ａｃｔｉｖｉｔｙ　ｓｃｏｒｅ（ＮＡＳ）とし、合計点数８点中、０～２点はＮＡＳＨではない、３～４点は判定保留、５点以上はＮＡＳＨと判断した。また、ｆｉｂｒｏｓｉｓ（線維化）の評価も行った。 [Evaluation of liver lesions]
All blindness was assessed by the pathologist, who performed grouping down. The diagnostic criteria were in accordance with the diagnostic criteria of "NASH Clinical Research Network Scoring System" proposed by Kleiner et al. This is a method for scoring the degree of NASH by scoring steatosis (fat deposition), lobular inflammation (intralobal inflammation), and hepatocyte ballooning (balloon-like change of hepatocytes) according to the classification shown in Table 1 above. Furthermore, three categories of fat deposition, intralobular inflammation, balloon-like changes of hepatocytes are combined to form NAFLD activity score (NAS), and 0 to 2 points are not NASH out of a total of 8 points, 3 to 4 points are judged Withheld, it was judged that NASH was more than 5 points. In addition, evaluation of fibrosis was also performed.
（５）肝線維化マーカー（Ｃｏｌ１ａ１、Ｔｇｆ－β１）
　凍結保存していた肝臓組織からＲＮＡｉｓｏ　Ｐｌｕｓ（タカラバイオ，滋賀）を用いて説明書通りの方法で、総ＲＮＡを抽出した。総ＲＮＡとＲｅｖｅｒＴｒａ　Ａｃｅ　ｑＰＣＲ　ＲＴ　Ｍａｓｔｅｒ　Ｍｉｘ（東洋紡、大阪）を用いて逆転写反応を行い、ｃＤＮＡを作成し、Ｒｅａｌ－ｔｉｍｅ　ＰＣＲに用いた。ＰＣＲはＬｉｇｈｔＣｙｃｌｅｒ　Ｎａｎｏ（ロシュ・ダイアグノスティックス、東京、日本）あるいはＳＴＥＰ　Ｏｎｅ　ｒｅａｌ－ｔｉｍｅ　ＰＣＲ　ｓｙｓｔｅｍ　（Ａｐｐｌｉｅｄ　Ｂｉｏｓｙｓｔｅｍｓ，Ｃａｒｌｓｂａｄ，ＣＡ，ＵＳＡ）を使用し、ＰＣＲの反応液の調製には、ＴＨＵＮＤＥＲＢＩＲＤ　ＳＹＢＲ　ｑＰＣＲ　Ｍｉｘ（東洋紡）を用いた。プライマーの合成はＦＡＳＭＡＣ（神奈川）に依頼し、購入した。ＰＣＲ反応は説明書通りの標準的なプログラムで実施した。各目的遺伝子（Ｃｏｌ１ａ１、Ｔｇｆ－β１）の発現量の相対比はβ－ａｃｔｉｎ遺伝子を内部標準として、比較Ｃｔ法で解析した。 (5) Liver fibrosis marker (Col1a1, Tgf-β1)
Total RNA was extracted from cryopreserved liver tissue using RNAiso Plus (Takara Bio, Shiga) according to the method described in the manual. Reverse transcription reaction was performed using total RNA and ReverTra Ace qPCR RT Master Mix (Toyobo, Osaka), and cDNA was prepared and used for Real-time PCR. PCR is carried out using LightCycler Nano (Roche Diagnostics, Tokyo, Japan) or STEP One real-time PCR system (Applied Biosystems, Carlsbad, CA, USA), and THUNDERBIRD SYBR qPCR is used to prepare a PCR reaction solution. Mix (Toyobo) was used. Primer synthesis was requested by FASMAC (Kanagawa) and purchased. The PCR reaction was carried out with a standard program as described. The relative ratio of the expression level of each target gene (Col1a1, Tgf-β1) was analyzed by the comparative Ct method using the β-actin gene as an internal standard.
（６）統計処理
　各項目の分析結果は、平均値±標準誤差（ＳＥ）で示し、統計解析にはＳＰＳＳ　ｓｔａｔｉｓｔｉｃｓ　２１（日本アイ・ビー・エム株式会社）を用いた。６群間の有意差の検定には一元配置分散分析およびＳｃｈｅｆｆｅ法（多重比較）、クロス集計はＦｉｓｈｅｒの直接確率の有意性検定にて行った。すべての検定で有意確率Ｐ＜０．０５を統計学的に有意差があると判断した。 (6) Statistical processing The analysis result of each item is shown as mean value ± standard error (SE), and SPSS statistics 21 (Japan IDM Ltd.) was used for statistical analysis. One-way analysis of variance and the Scheffe method (multiple comparisons) were used to test for significant differences among the six groups, and cross-tabulation was performed using Fisher's test of direct probability for significance. The significance probability P <0.05 was determined to be statistically significant in all tests.
〔実験結果〕
　Ｂ６マウスのうちＮｏｒｍａｌ食を摂取した群をＢ６Ｎ群、コレステロール１．２５質量％添加高脂肪飼料を摂取した群をＢ６ＬＣ群、コレステロール２．５質量％添加高脂肪飼料を摂取した群をＢ６ＨＣ群と表記する。同様にＡ／ＪマウスもＡ／ＪＮ群、Ａ／ＪＬＣ群、Ａ／ＪＨＣ群と表記する。 〔Experimental result〕
Of the B6 mice, a group receiving a normal diet is B6 N, a group receiving a high fat diet supplemented with 1.25 mass% cholesterol is a B6 LC group, and a group receiving a high fat diet supplemented with 2.5 mass% cholesterol a B6 HC group write. Similarly, A / J mice are also referred to as A / JN group, A / JLC group, and A / JHC group.
（１）血清・肝臓生化学検査値
　図１は血清コレステロール値を、図２は血清ＡＬＴ値を、図３は肝臓ＴＧ値を示す。
　本発明の飼料の摂取により、肝臓中のトリグリセリド量が有意に増加した（図３）。また、本発明の飼料の摂取により、Ｂ６マウス及びＡ／Ｊマウスの血清コレステロール値およびＡＬＴ値が有意に増加した（図１及び２）。 (1) Serum-liver biochemical test values FIG. 1 shows serum cholesterol level, FIG. 2 shows serum ALT level, and FIG. 3 shows liver TG level.
The intake of the feed of the present invention significantly increased the amount of triglyceride in the liver (FIG. 3). Also, intake of the feed of the present invention significantly increased serum cholesterol and ALT levels of B6 mice and A / J mice (FIGS. 1 and 2).
〔肝組織学的所見（肝病変の評価）〕
　結果を下記表４及び図４に示す。表４内の数値は個体数を示す。 [Hepatic findings (evaluation of liver lesions)]
The results are shown in Table 4 below and FIG. The numerical values in Table 4 indicate the number of individuals.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
　Ｂ６ＬＣ群、Ｂ６ＨＣ群、Ａ／ＪＬＣ群、及びＡ／ＪＨＣ群の肝組織所見で、ＮＡＦＬＤ　ａｃｔｉｖｉｔｙ　ｓｃｏｒｅが５点以上の個体が認められ（表４及び図４）、ＮＡＳＨと診断される個体が得られた。Ｂ６マウスと比較してＡ／Ｊマウスの方がＮＡＦＬＤ　ａｃｔｉｖｉｔｙ　ｓｃｏｒｅが高いマウスが多く、Ａ／ＪマウスはＮＡＳＨ易発性を有する、又はＡ／Ｊマウスは本実験で用いた飼料成分に対する感受性が高いことが確認された。 Liver histopathology of B6LC group, B6HC group, A / JLC group, and A / JHC group showed individuals with NAFLD activity score of 5 or more (Table 4 and Figure 4), and individuals diagnosed with NASH were obtained It was done. There are more mice with higher NAFLD activity score in A / J mice than in B6 mice, A / J mice have NASH susceptibility, or A / J mice have sensitivity to feed components used in this experiment. It was confirmed to be high.
〔肝線維化〕
　肝線維化所見がみられることはその予後の悪化の報告があることから注意すべき病変である。本実験において、ステージ２である中等度の線維化病変が確認された（図４）。線維化はＢ６及びＡ／Ｊマウスの双方において、コレステロール濃度依存的に悪化を示した。また、Ｂ６マウスと比較してＡ／Ｊマウスの方が高度な線維化病変を示した。Ａ／Ｊマウスにおいて肝臓におけるコレステロール蓄積はＢ６マウスに比べて高いレベルであり、これがＡ／Ｊマウスにおける高度な肝線維化に関与している可能性が考えられる。 [Liver fibrosis]
The presence of liver fibrosis findings is a lesion that should be noted as there has been a report of its prognostic deterioration. In this experiment, moderate fibrotic lesions at stage 2 were identified (FIG. 4). Fibrosis showed cholesterol-dependent deterioration in both B6 and A / J mice. In addition, A / J mice showed higher fibrotic lesions as compared to B6 mice. Cholesterol accumulation in the liver in A / J mice is at a higher level than in B6 mice, which may be responsible for high liver fibrosis in A / J mice.
〔肝線維化マーカー〕
　肝線維化マーカーである各ｍＲＮＡ（Ｃｏｌ１ａ１、Ｔｇｆ－β１）の発現量はＢ６マウス及びＡ／ＪマウスのＬＣ群及びＨＣ群において、各Ｎｏｒｍａｌ群と比較して有意に増加し又は増加傾向を示した（図５及び６）。 [Liver fibrosis marker]
The expression level of each mRNA (Col1a1, Tgf-β1) which is a liver fibrosis marker shows a significant increase or a tendency to increase in LC group and HC group of B6 mouse and A / J mouse in comparison with each normal group (Figures 5 and 6).
　以上をまとめると、ＮＡＳＨ又は肝線維化を高度に誘導する点で、飼料中のコレステロール含量を増やし、又はＡ／Ｊマウスを用いることが好ましい。 To summarize the above, it is preferable to increase the cholesterol content in the feed or to use A / J mice in that it highly induces NASH or liver fibrosis.
〔身体所見〕
　全ての群の総摂取エネルギー量は同等で（図７）、各系統内においてＮｏｒｍａｌ群とコレステロール負荷群（ＬＣ，ＨＣ）の体重に有意な差はみられなかったが（図８）、Ｂ６マウス及びＡ／ＪマウスのＬＣ群及びＨＣ群において体重当たりの肝臓重量の有意な増加が確認された（図９）。 [Physical findings]
Although total energy intake by all groups was equal (Fig. 7), there was no significant difference in body weight between Normal group and cholesterol loaded group (LC, HC) in each strain (Fig. 8), but B6 mice A significant increase in liver weight per body weight was confirmed in LC and HC groups of and A / J mice (FIG. 9).
２．試験例２
（１）実験方法
　８週齢雄性Ｃ５７ＢＬ／６Ｊマウス２２匹（日本ＳＬＣ）を購入し、初めの１週間は標準飼料（ＭＦ；オリエンタル酵母）を摂取させ飼育環境に順化させた。順化後の９週齢時から、標準飼料であるＭＦを摂取させた普通食群、ＭＦに粉末パーム油（商品名粉末油脂P-80；ナガセサンバイオ製）２５質量％、コレステロール（商品名コレステロール；和光純薬工業製）１．２５質量％及びコール酸ナトリウム塩（商品名コール酸ナトリウム；和光純薬工業製）０．２質量％を添加した飼料を摂取させたコール酸０．２％添加食群、ＭＦに粉末パーム油２５質量％、コレステロール１．２５質量％及びコール酸塩０．４質量％を添加したコール酸０．４％添加食群の３群各３～１１匹ずつに分け、２０週齢まで飼育した。食餌及び水は自由摂取とし、飼育期間中、体重測定を１週間に１回行った。
　すべての動物は、温度２４℃、湿度５５％、７～１９時は明期、１９～７時は暗期サイクルに設定された室内で飼育した。なお、本動物実験は「奈良女子大学動物実験に関する指針」ならびに「実験動物の飼育及び保管等に関する基準（昭和５５年３月総理府告示第６号）」に則して実施した。 2. Test example 2
(1) Experimental method Twenty-eight 8-week-old male C57BL / 6J mice (Japan SLC) were purchased, and a standard diet (MF; Oriental yeast) was ingested for the first week to acclimate to the breeding environment. Normal food group fed MF, which is a standard feed, from 9 weeks of age after acclimatization, to 25% by mass of powdered palm oil (trade name: powdered oil and fat P-80; manufactured by Nagasesan Bio), and cholesterol (trade name: MF) Cholesterol: 0.2% cholic acid ingested feed containing 1.25% by weight Wako Pure Chemical Industries, Ltd. and 0.2% by weight cholic acid sodium salt (trade name sodium cholate; Wako Pure Chemical Industries) 0.2% by weight Added food group, 3 to 3 each of 3 groups of 3 groups of cholic acid 0.4% added food group to which 25% by mass powdered palm oil, 1.25% by mass cholesterol and 0.4% by mass cholate were added to MF Separated and kept until 20 weeks old. Food and water were free intake, and body weight was measured once a week during the breeding period.
All animals were kept in a room set at a temperature of 24 ° C., a humidity of 55%, a light period of 7-19 o'clock and a dark cycle of 19-7 o'clock. The present animal experiment was conducted in accordance with "Guideline on animal experiment of Nara Women's University" and "Standard for breeding and storage of experimental animal (announcement No. 6 of the Prime Minister's notice in March 1959)".
（２）実験結果
　同週齢群間で有意差がなかったことから、マウスに与える飼料のコール酸の含有量がいずれであっても体重減少が見られず、ＮＡＦＬＤモデルマウスが得られることが確認された（図１０）。
　さらに、マウスに与える飼料のコール酸の含有量が少ないほど、体重が増加したマウス、すなわち優れたＮＡＦＬＤモデルマウスが得られることが確認された（図１０）。 (2) Experimental Results As there was no significant difference between the same age groups, no weight loss was observed regardless of the cholic acid content of the feed given to mice, and it was possible to obtain a NAFLD model mouse It was confirmed (Figure 10).
Furthermore, it was confirmed that the less the cholate content of the feed fed to mice, the more weight gaining mice, ie, the superior NAFLD model mice can be obtained (FIG. 10).
３．試験例３
（１）実験方法
　４週齢雄性Ｃ５７ＢＬ／６Ｊマウス２２匹（日本ＳＬＣ）を購入し、初めの１週間は標準飼料（ＭＦ；オリエンタル酵母）を摂取させ飼育環境に順化させた。順化後の５週齢時から、標準飼料であるＭＦを摂取させた普通食群、ＭＦに粉末パーム油（商品名粉末油脂P-80；ナガセサンバイオ製）２５質量％、コレステロール（商品名コレステロール；和光純薬工業製）１．２５質量％及びコール酸ナトリウム塩（商品名コール酸ナトリウム；和光純薬工業製）０．１質量％を添加した飼料を摂取させたコール酸０．１％添加食群、ＭＦに粉末パーム油２５質量％、コレステロール１．２５質量％及びコール酸塩０．４質量％を添加したコール酸０．４％添加食群の３群各３～１１匹ずつに分け、２０週齢まで飼育した。食餌及び水は自由摂取とし、飼育期間中、体重測定を１週間に１回行った。
　すべての動物は、温度２４℃、湿度５５％、７～１９時は明期、１９～７時は暗期サイクルに設定された室内で飼育した。なお、本動物実験は「奈良女子大学動物実験に関する指針」ならびに「実験動物の飼育及び保管等に関する基準（昭和５５年３月総理府告示第６号）」に則して実施した。 3. Test Example 3
(1) Experimental method Twenty-four 4-week-old male C57BL / 6J mice (Japan SLC) were purchased, and a standard diet (MF; Oriental yeast) was ingested for the first week to acclimate to the breeding environment. Normal food group fed MF, which is a standard feed, from 5 weeks of age after acclimatization, 25% by mass powdered palm oil (trade name: Powdered oil and fat P-80; manufactured by Nagasesan Bio) in MF, cholesterol (trade name) Cholesterol: 0.1% cholic acid ingested feed supplemented with 1.25% by mass Wako Pure Chemical Industries, Ltd. and 0.1% by mass sodium cholate (trade name sodium cholate; manufactured by Wako Pure Chemical Industries) Added food group, 3 to 3 each of 3 groups of 3 groups of cholic acid 0.4% added food group to which 25% by mass powdered palm oil, 1.25% by mass cholesterol and 0.4% by mass cholate were added to MF Separated and kept until 20 weeks old. Food and water were free intake, and body weight was measured once a week during the breeding period.
All animals were kept in a room set at a temperature of 24 ° C., a humidity of 55%, a light period of 7-19 o'clock and a dark cycle of 19-7 o'clock. The present animal experiment was conducted in accordance with "Guideline on animal experiment of Nara Women's University" and "Standard for breeding and storage of experimental animal (announcement No. 6 of the Prime Minister's notice in March 1959)".
（２）実験結果
　同週齢群間で有意差がなかったことから、マウスに与える飼料のコール酸の含有量がいずれであっても体重減少が見られず、ＮＡＦＬＤモデルマウスが得られることが確認された（図１１）。
　さらに、コール酸添加量が異なる飼料を５週齢時から１５週間摂取させると、すなわち、試験例２と比較して飼料投与開始時期を早めると、又は飼料投与期間を長くすると、より体重が増加したマウス、すなわちより優れたＮＡＦＬＤモデルマウスが得られることが確認された（図１１）。 (2) Experimental Results As there was no significant difference between the same age groups, no weight loss was observed regardless of the cholic acid content of the feed given to mice, and it was possible to obtain a NAFLD model mouse It was confirmed (Figure 11).
Furthermore, when a feed different in cholic acid addition amount is taken from 5 weeks of age to 15 weeks, that is, when the feed administration start time is advanced compared to Test Example 2, or when the feed administration period is extended, the weight gain is further increased. It was confirmed that a better NAFLD model mouse could be obtained (FIG. 11).
４．試験例４
（１）実験方法
　６週齢雄性Ｔｓｕｍｕｒａ－Ｓｕｚｕｋｉ　ｎｏｎ－ｏｂｅｓｅ（ＴＳＮＯ）マウス２４匹及びＴｓｕｍｕｒａ－Ｓｕｚｕｋｉ　ｏｂｅｓｅ　ｄｉａｂｅｔｅｓ（ＴＳＯＤ）マウス１２匹（いずれも動物繁殖研究所）を入手し、標準飼料（ＭＦ；オリエンタル酵母）を摂取させた普通食（ＮＭ）群、ＭＦに粉末パーム油２８．７５質量％、コレステロール１．２５質量％及びコール酸ナトリウム塩０．５質量％を添加した飼料を摂取させた高脂肪・コレステロール食（ＨＦＣ）群に分けた。飼料摂取開始後、３ヶ月及び６ヶ月時に屠殺した。各群６匹ずつとし、食餌及び水は自由摂取とした。本実験は動物繁殖研究所に委託し、飼料はオリエンタル酵母に作成を依頼した。本試験を実施するに当たり、各法令および指針の遵守はもとより、一般財団法人動物繁殖研究所の実験動物福祉規程および標準操作手順書に従い、動物福祉の精神に則り実施した。
　二酸化炭素吸入麻酔下で開腹処置を行い、シリンジを用いて腹部大静脈から採血した。直ちに肝臓を摘出し、０．９％(ｗ／ｖ)食塩水で洗浄後重量を測定した。肝臓の一部を切除し、１０％(ｗ／ｖ)中性ホルマリン液で固定した。
　試験例１に記載の血清・肝臓生化学的測定、病理組織学検討、及び肝線維化マーカーの欄に記載の方法にて、血清中のＡＬＴ、肝組織学的所見、肝線維化マーカー（Ｃｏｌ１ａ１、Ｔｇｆ－β１）を分析した。各目的遺伝子（Ｃｏｌ１ａ１、Ｔｇｆ－β１）の発現量の相対比はｇｌｙｃｅｒａｌｄｅｈｙｄｅ　３－ｐｈｏｓｐｈａｔｅ　ｄｅｈｙｄｒｏｇｅｎａｓｅ（Ｇａｐｄｈ）遺伝子を内部標準として、比較Ｃｔ法で解析した。また、体重測定を２又は４週間に１回行った。
　各項目の分析結果は、平均値±標準誤差（ＳＥ）で示した。群間の有意差の検定は統計解析ソフトＧｒａｐｈＰａｄ　Ｐｒｉｓｍ　７．０２を用いて、一元配置分散分析およびＢｏｎｆｅｒｒｏｎｉ法（多重比較）にて行い、有意確率Ｐ＜０．０５を統計学的に有意差があると判断した。 4. Test Example 4
(1) Experimental method Twenty six-week-old male Tsumura-Suzuki non-obese (TSNO) mice and 12 Tsumura-Suzuki obese diabetes (TSOD) mice (all at the Animal Reproductive Research Institute) were obtained, and a standard diet (MF; Oriental yeast), 28.75 wt% powdered palm oil, 1.25 wt% cholesterol and 0.5 wt% cholate sodium salt added to MF It was divided into fat and cholesterol diet (HFC) groups. The animals were sacrificed at 3 months and 6 months after the start of feed intake. Each group had 6 animals, and food and water were freely available. The experiment was commissioned to the Animal Breeding Research Institute, and the feed was requested to be produced by Oriental yeast. In carrying out this test, in addition to compliance with each law and guideline, in accordance with the Experimental Animal Welfare Regulations and Standard Operating Procedure Manual of the General Research Institute for Animal Breeding, it was conducted in accordance with the spirit of animal welfare.
The laparotomy was performed under carbon dioxide inhalation anesthesia, and blood was collected from the abdominal vena cava using a syringe. The liver was immediately removed, washed with 0.9% (w / v) saline and weighed. A part of the liver was excised and fixed with 10% (w / v) neutral formalin solution.
ALT in serum, liver histology findings, liver fibrosis marker (Col1a1) by the method described in the column of serum / liver biochemical measurement, histopathological examination, and liver fibrosis marker described in Test Example 1 , Tgf-β1). The relative ratio of the expression level of each target gene (Col1a1, Tgf-β1) was analyzed by the comparative Ct method using a glyceroldede 3-phosphate dehydrogenase (Gapdh) gene as an internal standard. In addition, weight measurement was performed once every two or four weeks.
The analysis results of each item are shown as mean value ± standard error (SE). Tests for significant differences between groups are performed by one-way analysis of variance and Bonferroni's method (multiple comparisons) using the statistical analysis software GraphPad Prism 7.02, and significant differences P <0.05 are statistically significant. I judged that there was.
（２）実験結果
　ＴＳＮＯマウスにおけるＮＭ群及びＨＦＣ群の３ヶ月時屠殺群、並びにＴＳＯＤマウスにおけるＨＦＣ群の３ヶ月時屠殺群をそれぞれＮＭ－ＴＳＮＯ－３ｍ、ＨＦＣ－ＴＳＮＯ－３ｍ、ＨＦＣ－ＴＳＯＤ－３ｍと表記し、ＴＳＮＯマウスにおけるＮＭ群及びＨＦＣ群の６ヶ月時屠殺群、並びにＴＳＯＤマウスにおけるＨＦＣ群の６ヶ月時屠殺群をそれぞれＮＭ－ＴＳＮＯ－６ｍ、ＨＦＣ－ＴＳＮＯ－６ｍ、ＨＦＣ－ＴＳＯＤ－６ｍと表記する。 (2) Experimental results NM group and HFC group 3-month sacrifice group in TSNO mice, and 3-month sacrifice group of HFC group in TSOD mice NM-TSNO-3 m, HFC-TSNO-3 m, HFC-TSOD- Marked as 3 m, 6-month sacrifice group of NM group and HFC group in TSNO mice and 6-month sacrifice group of HFC group in TSOD mice are respectively NM-TSNO-6m, HFC-TSNO-6m, HFC-TSOD- It is written as 6m.
〔血清・肝臓生化学検査値〕
　図１２は血清ＡＬＴ値の結果を示す。
　図１２から明らかなように、本発明の飼料の３ヶ月間又は６ヶ月間の摂取により、ＴＳＮＯ及びＴＳＯＤマウスの血清ＡＬＴ値が有意に増加した。 [Serum and liver biochemical test values]
FIG. 12 shows the results of serum ALT levels.
As apparent from FIG. 12, the intake of the feed of the present invention for 3 months or 6 months significantly increased the serum ALT value of TSNO and TSOD mice.
〔肝組織学的所見（肝病変・肝線維化の評価）〕
　図１３は肝組織学的所見の結果を示す。
　図１３から明らかなように、本発明の飼料の３ヶ月間又は６ヶ月間の摂取による、ＴＳＮＯマウスのＮＡＳＨ発症、さらには線維化病変が確認された。
　具体的には、ＨＦＣ群では軽度の脂肪変性、炎症及び風船様肝細胞がみられＮＡＳＨを発症していた。また、ＨＦＣ－ＴＳＮＯ－３ｍ群ではステージ１～２程度、ＨＦＣ－ＴＳＮＯ－６ｍ群ではステージ２～３程度の線維化病変がみられた。
　その一方で、ＴＳＯＤマウスではＮＡＳＨ発症及び線維化病変がみられたものの、ＨＦＣ－ＴＳＯＤ－３ｍ群ではステージ０～１程度、ＨＦＣ－ＴＳＮＯ－６ｍ群ではステージ１～２程度の線維化病変であった。
　以上をまとめると、肝線維化を高度に誘導する点で、ＴＳＯＤマウスよりＴＳＮＯマウスを用いることが好ましい。
　食事誘導肝線維化モデル動物としては、本モデルは線維化の程度はかなり強く、進行したＮＡＦＬＤを発症する優れたモデル動物であると言える。 [Hepatic findings (evaluation of liver lesions and liver fibrosis)]
FIG. 13 shows the results of liver histological findings.
As apparent from FIG. 13, NASH onset and further fibrotic lesions in TSNO mice were confirmed by intake for 3 or 6 months of the feed of the present invention.
Specifically, mild steatosis, inflammation and balloon-like hepatocytes were observed in the HFC group and NASH was developed. In addition, fibrotic lesions were observed in stages 1 to 2 in the HFC-TSNO-3m group and in stages 2 to 3 in the HFC-TSNO-6m group.
On the other hand, although NASH onset and fibrotic lesions were observed in TSOD mice, fibrotic lesions were about 0-1 stage in the HFC-TSOD-3m group and about 1-2 stages in the HFC-TSNO-6m group. The
In summary, it is preferable to use TSNO mice rather than TSOD mice in terms of highly inducing liver fibrosis.
As a diet-induced liver fibrosis animal model, this model has a fairly high degree of fibrosis and can be said to be an excellent model animal to develop advanced NAFLD.
〔肝線維化マーカー〕
　図１４及び１５は肝線維化マーカーの結果を示す。
　図１４及び１５から明らかなように、本発明の飼料摂取によるＴＳＮＯマウスの肝線維化マーカーの有意な増加が確認された。
　具体的には、肝臓における各ｍＲＮＡ（Ｃｏｌ１ａ１、Ｔｇｆ－β１）の発現量はＴＳＮＯマウスのＨＦＣ群において、各ＮＭ群と比較して有意に増加した。 [Liver fibrosis marker]
Figures 14 and 15 show the results of liver fibrosis markers.
As apparent from FIGS. 14 and 15, a significant increase in liver fibrosis marker in TSNO mice was confirmed by the feed intake of the present invention.
Specifically, the expression level of each mRNA (Col1a1, Tgf-β1) in the liver was significantly increased in the HFC group of TSNO mice as compared to each NM group.
〔身体所見〕
　図１６は肝臓重量／体重比の結果を、図１７は体重の推移をそれぞれ示す。
　図１６から明らかなように、本発明の飼料摂取によるＴＳＮＯ及びＴＳＯＤマウスの肝臓重量／体重比の有意な増加が確認された。
　さらに具体的には、ＨＦＣ－ＴＳＮＯ群では、同週齢ＮＭ－ＴＳＮＯ及びＨＦＣ－ＴＳＯＤ群に比較して、肝臓重量／体重比が高値を示した。
　尚、体重はＮＭ－ＴＳＮＯ－３ｍ及びＨＦＣ－ＴＳＮＯ－３ｍ間に有意な差はみられなかった（図１７）。 [Physical findings]
FIG. 16 shows the result of liver weight / body weight ratio, and FIG. 17 shows the transition of weight.
As apparent from FIG. 16, a significant increase in liver weight / body weight ratio of TSNO and TSOD mice was confirmed by the feed intake of the present invention.
More specifically, in the HFC-TSNO group, the liver weight / body weight ratio showed a high value as compared with the same age NM-TSNO and HFC-TSOD groups.
There was no significant difference in body weight between NM-TSNO-3m and HFC-TSNO-3m (FIG. 17).
５．試験例５
（１）実験方法
　４及び８週齢雄性Ｃ５７ＢＬ／６Ｊマウス２６匹（日本ＳＬＣ）を購入し、初めの１週間は標準飼料（ＭＦ；オリエンタル酵母）を摂取させ飼育環境に順化させた。順化後の５及び９週齢時から、標準飼料であるＭＦを摂取させた普通食群、ＭＦに粉末パーム油（商品名粉末油脂Ｐ－８０；ナガセサンバイオ製）２５質量％、コレステロール（商品名コレステロール；和光純薬工業製）１．２５質量％及びコール酸ナトリウム塩（商品名コール酸ナトリウム；和光純薬工業製）０．１質量％を添加した飼料を摂取させたコール酸０．１％添加食群、ＭＦに粉末パーム油２５質量％、コレステロール１．２５質量％及びコール酸ナトリウム塩０．２質量％を添加したコール酸０．２％添加食群、ＭＦに粉末パーム油２５質量％、コレステロール１．２５質量％及びコール酸ナトリウム塩０．４質量％を添加したコール酸０．４％添加食群の４群各３～５匹ずつに分け、１６又は２４週間飼育した。食餌及び水は自由摂取とし、飼育期間中、体重測定を１週間に１回行った。
　すべての動物は、温度２４℃、湿度５５％、７～１９時は明期、１９～７時は暗期サイクルに設定された室内で飼育した。なお、本動物実験は「奈良女子大学動物実験に関する指針」ならびに「実験動物の飼育及び保管等に関する基準（昭和５５年３月総理府告示第６号）」に則して実施した。 5. Test Example 5
(1) Experimental Method Twenty-six male 4- and 8-week-old male C57BL / 6J mice (Japan SLC) were purchased, and a standard diet (MF; Oriental yeast) was ingested for the first week to acclimate to the breeding environment. A normal diet group ingested MF, which is a standard diet, from 5 and 9 weeks of age after acclimatization, 25% by mass of powdered palm oil (trade name Powdered oil and fat P-80; manufactured by Nagasesan Bio) in MF, cholesterol (MF) Trade name: cholesterol; Wako Pure Chemical Industries, Ltd. 1.25% by mass Cholic acid sodium salt (trade name: sodium cholate; Wako Pure Chemical Industries, Ltd.) 0.1% by mass Cholic acid ingested with feed added thereto. 1% added food group, 25% by mass powdered palm oil, 1.25% by mass cholesterol and 0.2% by mass cholic acid sodium salt added to caffeic acid 0.2% added food group with MF, powdered palm oil 25 to MF Each group was divided into 3 to 5 animals each in 4 groups of the cholic acid 0.4% -added food group to which mass%, cholesterol 1.25% by mass, and cholic acid sodium salt 0.4% by mass were added, and reared for 16 or 24 weeks. Food and water were free intake, and body weight was measured once a week during the breeding period.
All animals were kept in a room set at a temperature of 24 ° C., a humidity of 55%, a light period of 7-19 o'clock and a dark cycle of 19-7 o'clock. The present animal experiment was conducted in accordance with "Guideline on animal experiment of Nara Women's University" and "Standard for breeding and storage of experimental animal (announcement No. 6 of the Prime Minister's notice in March 1959)".
　屠殺前に８時間絶食させたのち、イソフルラン吸入麻酔下で開腹処置を行い、シリンジを用いて心臓から採血した。直ちに肝臓、睾丸周囲脂肪を摘出し、０．９％(ｗ／ｖ)食塩水で洗浄後重量を測定した。肝臓の一部を切除し、１０％(ｗ／ｖ)中性ホルマリン液で固定した。 The animals were fasted for 8 hours before sacrifice, then underwent laparotomy under isoflurane inhalation anesthesia, and blood was collected from the heart using a syringe. Immediately, the liver and peritesticular fat were removed, and after washing with 0.9% (w / v) saline, the weight was measured. A part of the liver was excised and fixed with 10% (w / v) neutral formalin solution.
　〔血清グルコース値〕
　ムタロターゼ・ＧＯＤ法を用いた測定キットであるグルコースＣＩＩ－テストワコー（和光純薬工業）を使用し、説明書通りの方法で血清中のグルコースの測定を行った。
　測定は１検体につき２回行い、その平均値を測定した。
　〔血清インスリン値〕
　ＥＬＩＳＡ法を用いた測定キットであるマウスインスリン測定キット（森永生科学研究所）を使用し、説明書通りの方法で血清中のインスリンの測定を行った。
　測定は１検体につき２回行い、その平均値を測定した。
　〔インスリン抵抗性指標〕
　インスリン抵抗性指標は血清インスリン濃度および血清グルコース濃度を用い、次式により求めた。なお、本実験では国際単位の代わりに血清インスリン濃度（ng/mL）を用いて算出し、インスリン抵抗性の相対的な強弱を比較することで検討を行った。
インスリン抵抗性指標 ＝ 血清インスリン値 (ng/mL) × 血清グルコース値 (mg/dL) ÷ ４０５ [Serum glucose level]
Using glucose CII-Test Wako (Wako Pure Chemical Industries), which is a measurement kit using the mutarotase-GOD method, glucose in serum was measured according to the method described in the instruction.
The measurement was performed twice for one sample, and the average value was measured.
Serum insulin level
Using the mouse insulin measurement kit (Morinaga Institute of Science), which is a measurement kit using the ELISA method, insulin in serum was measured according to the method described in the instruction.
The measurement was performed twice for one sample, and the average value was measured.
[Insulin resistance index]
The insulin resistance index was determined by the following equation using serum insulin concentration and serum glucose concentration. In addition, in this experiment, it calculated using serum insulin concentration (ng / mL) instead of an international unit, and examined by comparing the relative strength of insulin resistance.
Insulin resistance index = serum insulin level (ng / mL) × serum glucose level (mg / dL) 405 405
　〔血清中のＡＬＴ、肝臓トリグリセリド、肝組織学的所見、肝線維化マーカー（Ｃｏｌ１ａ１、Ｔｇｆ－β１）〕
　試験例１に記載の血清・肝臓生化学的測定、病理組織学検討、及び肝線維化マーカーの欄に記載の方法にて、血清中のＡＬＴ、肝臓トリグリセリド、肝組織学的所見、肝線維化マーカー（Ｃｏｌ１ａ１、Ｔｇｆ－β１）を分析した。各目的遺伝子（Ｃｏｌ１ａ１、Ｔｇｆ－β１）の発現量の相対比はＧａｐｄｈ遺伝子を内部標準として、比較Ｃｔ法で解析した。 [ALT in serum, liver triglyceride, liver histology, liver fibrosis marker (Col1a1, Tgf-β1)]
ALT in serum, liver triglyceride, liver histopathological findings, liver fibrosis by the method described in the column of serum / liver biochemical measurement, histopathological examination, and liver fibrosis marker described in Test Example 1 Markers (Col1a1, Tgf-β1) were analyzed. The relative ratio of the expression level of each target gene (Col1a1, Tgf-β1) was analyzed by the comparative Ct method using the Gapdh gene as an internal standard.
　各項目の分析結果は、平均値±標準誤差（ＳＥ）で示した。同週齢３群間及び２群間の有意差の検定には一元配置分散分析及びＢｏｎｆｅｒｒｏｎｉ法、並びに対応のないt検定を用い、有意確率Ｐ＜０．０５を統計学的に有意差があると判断した。 The analysis results of each item are shown as mean value ± standard error (SE). There is a statistically significant difference in significance probability P <0.05 between the 3 weeks of age group and the 2 groups using the one-way analysis of variance and Bonferroni method and the unpaired t test I judged.
（２）実験結果
〔血清・肝臓生化学検査値〕
　表５は血清ＡＬＴ値、血清グルコース値、血清インスリン値及びインスリン抵抗指標の結果を示す。 (2) Experimental results [serum and liver biochemical test values]
Table 5 shows the results of serum ALT level, serum glucose level, serum insulin level and insulin resistance index.
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
　表５から明らかなように、本発明の飼料摂取により、Ｃ５７ＢＬ／６Ｊマウスの血清ＡＬＴ値が有意に増加した。
　特に、コール酸０．４％添加食群では、コール酸０．２％添加食群に比較して、血清ＡＬＴ値が高値を示した。
　その一方で、コール酸０．２％添加食群では、コール酸０．４％添加食群に比較して、インスリン抵抗性指標が高値傾向を示した。インスリン抵抗性はヒトにおけるＮＡＳＨ病態で高頻度にみられることから、インスリン抵抗性指標が普通食摂取群より高値を示すことがより望ましく、コール酸添加濃度は０．４％より０．２％の方がより優れたＮＡＦＬＤモデル動物を作製できるといえる。 As apparent from Table 5, the feed intake of the present invention significantly increased the serum ALT value of C57BL / 6J mice.
In particular, in the cholic acid 0.4% -added food group, the serum ALT value was higher than that of the cholic acid 0.2% -added food group.
On the other hand, in the cholic acid 0.2% -added food group, the insulin resistance index tended to be higher compared to the cholic acid 0.4% -added food group. Since insulin resistance is frequently observed in NASH disease states in humans, it is more desirable that the insulin resistance index shows a higher value than the normal food intake group, and the cholic acid addition concentration is 0.2% than 0.4%. It can be said that a better NAFLD model animal can be produced.
〔肝組織学的所見（肝病変・肝線維化の評価）〕
　図１８は肝組織学的所見の結果を示す。
　図１８から明らかなように、本発明の飼料摂取によるＣ５７ＢＬ／６Ｊマウスの肝線維化が確認された。
　特に、コール酸０．４％添加食群において、脂肪変性及び高度な炎症がみられた。また、線維化は中心静脈周囲に極軽度にみられた。コール酸０．２％及び０．１％添加食群において、軽度～中等度の脂肪変性及び炎症がみられ、ＮＡＳＨの特徴的な所見がみられた。線維化は中心静脈周囲及び肝細胞周囲に軽度な病変（ステージ１）がみられた。 [Hepatic findings (evaluation of liver lesions and liver fibrosis)]
FIG. 18 shows the results of liver histological findings.
As apparent from FIG. 18, hepatic fibrosis in C57BL / 6J mice was confirmed by the feed intake of the present invention.
In particular, steatosis and severe inflammation were observed in the cholic acid 0.4% -added diet group. Also, fibrosis was very mild around the central vein. Mild to moderate steatosis and inflammation were seen in the 0.2% and 0.1% chow diet diet groups, with characteristic findings of NASH. Fibrosis was a mild lesion (stage 1) around the central vein and around hepatocytes.
〔肝線維化マーカー〕
　図１９及び２０は肝線維化マーカーの結果を示す。
　図１９及び２０から明らかなように、本発明の飼料摂取によるＣ５７ＢＬ／６Ｊマウスの肝線維化マーカーの有意な増加又は増加傾向が確認された。 [Liver fibrosis marker]
Figures 19 and 20 show the results of liver fibrosis markers.
As apparent from FIGS. 19 and 20, a significant increase or a tendency of a marker for fibrosis of liver of C57BL / 6J mice by the feed intake of the present invention was confirmed.
〔身体所見〕
　下記表６は体重及び睾丸周囲脂肪重量／体重比の結果を示す。
　表６から明らかなように、特に、コール酸添加濃度が０．１又は０．２質量％である飼料摂取群において、同週齢普通食群に比較して、体重及び睾丸周囲脂肪重量／体重比の増加が確認された。すなわち、コール酸添加濃度が０．４質量％未満（例えば０．１又は０．２質量％）である飼料を摂取させることにより、より優れたＮＡＦＬＤモデルマウスが得られることが確認された。 [Physical findings]
Table 6 below shows the results of body weight and testis surrounding fat weight / body weight ratio.
As apparent from Table 6, in particular, in the feed intake group in which the cholic acid addition concentration is 0.1 or 0.2% by mass, the body weight and testicular fat weight / body weight in comparison with the same-week-old normal diet group An increase in the ratio was confirmed. That is, it was confirmed that a better NAFLD model mouse can be obtained by feeding a feed having a cholic acid addition concentration of less than 0.4% by mass (for example, 0.1 or 0.2% by mass).
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
　本発明のモデル動物、その作製方法、及びそれを作製するための飼料は、ＮＡＦＬＤの病態又はメカニズムの解明、ＮＡＦＬＤに対する新規予防又は治療薬の開発等に有用である。 The model animal of the present invention, a method for producing the same, and a feed for producing the same are useful for elucidating the pathogenesis or mechanism of NAFLD, development of a novel prophylactic or therapeutic drug for NAFLD, and the like.

Claims (5)

  1. 　コール酸又はその塩、及びコレステロールを含有することを特徴とする非アルコ－ル性脂肪肝疾患モデルマウス作製用飼料。 A feed for producing a non-alcoholic fatty liver disease mouse model, which comprises cholic acid or a salt thereof, and cholesterol.
  2. 　標準飼料を含有することを特徴とする請求項１に記載の飼料。 The feed according to claim 1, which contains a standard feed.
  3. 　脂質エネルギー比率が３０％以上であることを特徴とする請求項１又は２に記載の飼料。 The feed according to claim 1 or 2, wherein the lipid energy ratio is 30% or more.
  4. 　マウスに請求項１～３のいずれかに記載の飼料を与えて飼育する工程を含むことを特徴とする非アルコ－ル性脂肪肝疾患モデルマウスの作製方法。 A method for producing a non-alcoholic fatty liver disease model mouse, comprising the step of feeding a mouse the feed according to any one of claims 1 to 3 and rearing the mouse.
  5. 　請求項４に記載の方法により作製された非アルコ－ル性脂肪肝疾患モデルマウス。 A non-alcoholic fatty liver disease mouse model prepared by the method according to claim 4.
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