JP2014209849A - NASH model animal - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a NASH model animal in which the pathology characteristic of NASH in humans are produced, and a method for producing the same.SOLUTION: The NASH model animal is obtained by causing the animal to ingest a high-fat diet and administering a liver damaging agent at least four times.

Description

  The present invention relates to a NASH model animal showing a pathological condition similar to human non-alcoholic steatohepatitis (NASH) and a method for producing the same.

  In recent years, an increasing number of obese people have no drinking habits but become fatty liver due to cirrhosis or liver cancer. Among fatty liver diseases, a case in which a fatty liver disorder similar to alcoholic liver disorder is observed despite no apparent alcohol consumption is called nonalcoholic fatty liver disease (NAFLD), of which hepatocytes in liver tissue A case in which steatohepatitis with balloon-like swelling is observed is particularly called NASH. Since the NASH has a high risk of becoming cirrhosis or liver cancer, development of a preventive and therapeutic means is desired.

  The development of a NASH therapeutic drug requires a model of the disease state. As model animals of NASH or NAFLD, methionine and / or choline-deficient diet models (Non-patent Document 1, Patent Document 1), nitrite and hydroxylamine are administered to cause methemoglobinemia, and hypoxia in vivo Although the model (patent document 2) etc. which form is known, these cannot be said to reflect the pathological condition of NASH histologically. In addition, a model mouse that administers an N-acetyl-β-D-glucosaminidase inhibitor such as streptozotocin (Patent Document 3), a model mouse that administers a high fat diet and a tetracycline antibiotic (Patent Document 4), a high fat diet intake And a model mouse (Non-patent Document 2) in which carbon tetrachloride is administered once.

JP 2006-349457 A WO2008 / 018191 JP 2009-178143 A WO2006 / 030792

Journal of Gastroenterology and Hepatology 23 (2008) 1635-1648 World Journal of Gastroenterology 2005; 11 (9): 1392-1395

However, the pathological findings of these animal models show only large fatty liver, and there is no inflammation around the central lobule vein and balloon-like swelling of hepatocytes, which is characteristic of NASH. This animal model is hardly a human NASH model.
Therefore, the subject of this invention is providing the NASH model animal which produces the pathological condition peculiar to human NASH, and its production method.

  Therefore, the present inventor has made various studies to produce a model animal exhibiting a pathological condition peculiar to human NASH. Surprisingly, a drug that induces hepatic disorder while ingesting a high-fat diet to obesity (hereinafter referred to as a “health disorder”) , A hepatic disorder drug), is a large drop of fatty liver, inflammation around the central vein in the leaflet of the liver, and hepatocytes are swollen like a balloon. The present inventors have found that a model animal having a specific pathological condition can be prepared, and that the use of the model animal can evaluate the NASH preventive and therapeutic effect of the test substance and can screen for NASH preventive and therapeutic agents.

Therefore, the present invention provides a NASH model animal obtained by ingesting a high fat diet and administering a hepatotoxic agent four times or more.
The present invention also provides a method for producing a NASH model animal, characterized by ingesting a high fat diet and administering a hepatic disorder agent four or more times.
The present invention also provides a screening method for a NASH prophylactic / therapeutic agent characterized by using the NASH model animal.
Furthermore, the present invention provides a method for evaluating the NASH preventive and therapeutic effect of a test substance, characterized by using the NASH model animal.

  The NASH model animal of the present invention is a large-scale fatty liver, has inflammation around the central vein in the lobule of the liver, and has the symptoms peculiar to human NASH that hepatocytes are swollen like a balloon. It is useful as a human NASH model. By using the NASH model animal of the present invention, it becomes possible to screen for drugs and foods effective for human NASH. It can also be used to evaluate NASH preventive and therapeutic effects of test substances.

It is a figure which shows the histological change of the hepatocyte by high fat diet of Example 1, and carbon tetrachloride (0.1 mL / kg body weight) administration. A: Group 1 (untreated control group), B: Group 2 (high fat diet control group), C: Group 3 (high fat diet + carbon tetrachloride once administration group), DF: Group 9 (high fat Diet + carbon tetrachloride 8 times administration group) A: Normal liver histology. B: Small fat droplets in hepatocytes observed from around the central vein to the middle leaflet zone. C: Small fat droplets observed from around the central vein to the middle leaflet zone as in the high fat diet control group. D: Large drop fatty liver (arrowhead). E: Balloon-like swelling of the hepatocytes observed around the central vein (arrow). F: Lymphocyte-dominated inflammatory cell infiltration around the central vein. *: Central vein, #: Portal region, A to D, F: × 20, E: × 40, A to F: HE staining It is a figure which shows the fibrosis by high fat diet of Example 1, and carbon tetrachloride (0.1 mL / kg body weight) administration. A: Group 1 (untreated control group), B: Group 2 (high fat diet control group), C: Group 3 (high fat diet + carbon tetrachloride single administration group), D: Group 9 (high fat diet + Carbon tetrachloride Eight times administration group) A: Histological image of normal liver. B, C: Fibrosis was not confirmed in both the central vein area and the portal vein area. D: Fibrosis was observed surrounding the hepatocytes around the central vein. *: Central vein, #: Portal vein region, A to D: x20, MT staining AC is a figure which shows the fibrosis by administration of the high fat diet of Example 2, and carbon tetrachloride (0.25 mL / kg body weight) 8 times. A: Large droplet fatty liver; large droplet fat accumulates in hepatocytes from the central part of the lobule to the peripheral part, B: vacuoles in the cytoplasm are positive for Sudan III staining (fatty), C: minor fibers around the central vein *: Central vein, #: portal vein area, A: × 10, B, C: × 20A: HE staining, B: Sudan III staining (frozen section), C: MT staining AB is a figure which shows the inflammatory symptom by 8 times administration of the high fat diet of Example 2 and carbon tetrachloride (0.25 mL / kg body weight). A: Infiltration of inflammatory cells around central vein, B: Balloon-like enlargement of hepatocytes observed around central vein (arrow) *: Central vein, A: × 20, B: × 40, A, B: HE staining

  The NASH model animal of the present invention can be prepared by ingesting a high fat diet and administering a hepatic disorder agent 4 times or more.

  The animal to be used is not particularly limited, and examples thereof include mice, rats, guinea pigs, hamsters, rabbits, monkeys, etc., but it is preferable to use mice. In the case of using a mouse, it may be a normal mouse and may be a hybrid or strain, but a mouse used as an experimental animal, for example, C57BL / 6 mouse, C3H mouse, ICR mouse, BALB / c line A mouse or the like is preferable.

  The high-fat diet may be a diet containing 20% by mass or more of fat, and a diet containing 30 to 80% by mass of fat, particularly a diet containing 40 to 60% by mass of fat is preferable. As fat, all kinds of fat can be used regardless of animal fat and vegetable fat, and examples thereof include corn oil, soybean oil, rapeseed oil, beef tallow, and lard. Pork fat is preferred. In addition, components other than fat in the high-fat diet may be normal dietary components, and may include proteins, carbohydrates, minerals, and the like.

  The intake period of the high fat diet is not particularly limited as long as it is necessary for obesity to occur. However, it is preferably 2 weeks or longer, more preferably 4 weeks or longer, and particularly preferably 8 to 16 weeks. Moreover, the start time of high fat diet intake is not particularly limited, but is preferably from 5 weeks of age. Moreover, what is necessary is just to let an animal ingest a high fat diet freely every day.

  The hepatotoxic agent used is not particularly limited, and examples thereof include carbon tetrachloride, galactosamine, acetaminophen, LPS (lipopolysaccharide), and carbon tetrachloride is particularly preferable.

  It is important that hepatotoxic agents be administered four or more times in order to cause animals to develop symptoms peculiar to human NASH. The number of administration is preferably 4 to 10 times, and more preferably 4 to 8 times. When the number of administrations is less than 4, no inflammation or fibrosis occurs in the liver, and symptoms unique to human NASH do not develop.

  The means for administering the hepatic disorder agent is not particularly limited, such as intraperitoneal administration, intravenous administration, oral administration, and subcutaneous administration, but subcutaneous administration is preferred. In addition, it is important for the dose to be lower than 1 ml / kg body weight / dose that causes normal liver damage in order to develop symptoms unique to human NASH. 0.05 to 0.5 mL / kg body weight / time is preferable, and 0.1 to 0.25 mL / kg body weight / time is more preferable. If it is less than 0.05 mL / kg body weight / time, it may not exhibit NASH symptoms, and if it is more than 0.5 mL / kg body weight / time, it does not develop symptoms peculiar to human NASH and normal liver damage. Since it may become a model animal, it is not preferable. The start time of administration of the hepatic disorder is not particularly limited, but is preferably 2 weeks after the start of high fat diet intake, more preferably 4 weeks after the start of high fat diet, and more preferably 8 weeks. Later. In addition, the administration interval of the hepatotoxic agent is preferably 1 to 3 times a week, particularly preferably twice a week. In addition, it is preferable to continue ingesting a high fat diet during the period of administration of the hepatopathy agent.

  More preferable high fat diet intake and hepatic disorder administration schedule is that the high fat diet is ingested for 8 weeks or more, then the high fat diet is continued and the hepatic disorder agent is 0.1 to 0 twice a week. 25 mL / kg body weight / dose, 4 times or more in total.

  If a high-fat diet is ingested and a hepatotoxic agent is administered four or more times, pathological findings peculiar to human NASH appear in animals. The pathological condition is macroscopic fatty liver, inflammation around the central vein in the leaflet of the liver, and hepatocytes swollen like balloons. In the conventional NASH model, none of the pathological findings of both inflammation and balloon-like enlargement around the central vein in the lobule and large droplet fatty liver occurred. Therefore, the NASH model animal of the present invention is particularly useful as a pathological model of human NASH. Here, the balloon-like swelling of the hepatocytes is not particularly limited in the place where the balloon-like swelling occurs, but it may occur around the central vein because it is more similar to the symptoms of human NASH. preferable. Fatty liver means a state in which a large amount of fat is deposited in the liver, and more specifically, a state in which large fatty liver (deposition) and / or small fat accumulation (deposition) is observed. However, large droplet fatty liver (deposition) needs to be observed in that it is more similar to human NASH.

  The NASH model animal of the present invention preferably also exhibits a symptom that the liver tissue becomes fibrotic, and the NASH model animal that has developed until fibrosis is known as Matteoni et al. The NASH can be a model animal of NASH with a more advanced pathological condition. In addition, it is more preferable that fibrosis occurs around the central vein because it is similar to the symptoms of human NASH.

  Furthermore, the NASH model animal of the present invention is selected from serum aspartate aminotransferase (AST (GOT)), alanine aminotransferase (ALT (GPT)), free fatty acid (NEFA), and neutral fat (TG) 1 The above is higher than that of animals bred with normal feed. Preferably, the AST is 200 U / L or more, more preferably 500 U / L or more, and still more preferably 1000 U / L or more. ALT is 300 U / L or more, more preferably 800 U / L or more, and still more preferably 1200 U / L or more. Free fatty acid (NEFA) is 1000 μEq / L or more, more preferably 1200 μEq / L or more. The neutral fat is 20 mg / dL or more, more preferably 30 mg / dL or more. In human NASH patients, it is reported that the above index is higher than that in healthy individuals, and the NASH model animal of the present invention shows a pathological condition similar to the symptoms of human NASH.

  The NASH model animal of the present invention exhibits symptoms specific to human NASH, and is useful for studying the pathology of NASH, as well as screening for NASH prophylactic and therapeutic agents (NASH prophylactic and therapeutic foods). Useful for. It is also useful for evaluating NASH preventive and therapeutic effects of test substances.

  By ingesting a high-fat diet in animals, administering a test substance before, during and / or after administration of a hepatotoxic agent, and administering the hepatotoxic agent four times or more, and then observing the degree of hepatic disorder Thus, the NASH preventive and therapeutic effect of the test substance can be evaluated, and a NASH preventive and therapeutic agent can be screened. Here, the degree of liver damage can be determined by comparison with a test substance non-administered group and / or with a normal animal group (a normal diet group, a hepatotoxic agent non-administered group, etc.).

  EXAMPLES Next, although an Example is given and this invention is demonstrated in detail, this invention is not limited to this at all.

Example 1
(1) Production of model animals The produced NASH model mice are shown below (Table 1).
Carbon tetrachloride (0.1 mL / kg body weight) was subcutaneously administered to male C57BL / 6N mice fed with a high-fat diet (40% beef tallow mixed feed) and fed for 8 weeks under the following conditions. After the last administration, the blood was sacrificed under ether anesthesia. In addition, livers were collected from 5 animals in each group and fixed with 10% neutral buffered formalin. Table 2 shows the composition of the high fat diet.
For Group 1 and Group 9, blood was collected from the abdominal aorta just prior to sacrifice. The obtained blood was left on ice for 1 hour or more, and then centrifuged at 3000 rpm for 15 minutes to prepare serum.
When carbon tetrachloride was administered twice or more, the maximum number of administrations per week was set to twice.

(2) Histopathological observation The liver after fixation was cut out by a conventional method, embedded in paraffin, and then the paraffin block was sliced to a thickness of about 4 μm, and hematoxylin-eosin (HE) staining was performed. Observed with a microscope. In addition, Masson's trichrome (MT) staining was performed on paraffin sections to confirm the presence or absence of collagen fibers.
(3) Measurement of blood biochemical properties Using a 7170 automatic analyzer (Hitachi Ltd.), aspartate aminotransferase (AST (GOT)), alanine aminotransferase (ALT (GPT)), free fatty acid (NEFA) ), Neutral fat (TG) was measured.
(4) Diagnostic criteria for NASH Currently, the classification of histopathological diagnosis of NASH in the clinic includes Matteononi et al. (Gastroenterology 1999, 116: 1413-1419), Kleiner et al. (Hepatology 2005, 41: 1313-1321). ) And Brunt et al. (American Journal of Gastroenterology 1999, 94: 2467-2474). In the American Topic Society Single Topic Conference 2002, the essential findings of NASH include large droplet fatty liver, intralobular inflammation, and balloon-like swelling of hepatocytes. 1419)) The above is histologically diagnosed as NASH (Table 3). The Japan Society for Hepatology also encourages the classification of Matteoni et al. Therefore, this time, the classification of NAFLD by Matteoni et al. Was used as a diagnostic criterion for NASH.
That is, NASH requires large droplet fatty liver, lobular inflammation and balloon-like swelling of hepatocytes (Type 3 in Table 3), and in addition to these symptoms, when Mallory bodies or fibrosis is observed It is classified as Type 4.
In addition, in clinical human NASH, it has been reported that intralobular inflammation, balloon-like swelling and / or fibrosis occur around the central vein, so the above symptoms occur around the central vein Moreover, it can be said that the pathological condition is more similar to the symptoms of human NASH.

(5) Results (a) Histopathological changes in the high-fat diet control group (Group 2) In the high-fat diet control group (Group 2), small fat droplets were observed in hepatocytes from around the central vein to the middle leaflet zone. (FIG. 1B). Since small fat droplets were not observed in the untreated control group, it was considered to be a change caused by administration of a high fat diet.

(B) Histopathological change with time after administration of high fat diet + carbon tetrachloride (Group 3-5)
In the high-fat diet + carbon tetrachloride once-administered group (Group 3-5), small fat droplets were observed in the middle leaflet zone from the central vein as in the high-fat diet control group (Group 2) (FIG. 1C). However, in these groups, findings such as balloon-like cells and fibrosis of large droplet fatty liver observed in NAFLD and hepatocytes observed in NASH were not confirmed (Tables 4, 5, and 1C). ).

(C) Histopathological change accompanying increase in the number of administrations of high fat diet + carbon tetrachloride (Group 6-9)
Large drop fatty liver was observed in the multiple-dose group (Group 6-9) of high fat diet + carbon tetrachloride (FIG. 1D). The large droplet fatty liver tended to become more frequent and stronger as the number of administrations of carbon tetrachloride increased (Tables 4 and 5). On the other hand, small fat droplets around the central vein tended to decrease (Tables 4 and 5). To date, there is no report on the relationship between large droplet fatty liver and small lipid droplets, but large droplet fatty liver was considered to be a change caused by multiple administrations of carbon tetrachloride. Furthermore, in the multiple administration group of carbon tetrachloride (Group 6-9), balloon-like swelling of hepatocytes was observed in each group at a ratio of 2-3 cases out of 5 around the central vein (FIG. 1E).

  Further, in the group (Group 7-9) administered with the high fat diet + carbon tetrachloride four times or more, inflammatory cell infiltration (inflammation) mainly consisting of lymphocytes and mild fibrosis were observed around the central vein ( FIG. 1F, FIG. 2D). The incidence of inflammatory cell infiltration (inflammation) tended to increase in proportion to the number of administrations (Tables 4 and 5). Fibrosis was observed around the central vein and surrounding hepatocytes (FIG. 2D), and was observed in each group at a rate of 3 to 4 out of 5 cases (Tables 4 and 5). However, fibrosis that progressed to bridging fibrosis as reported in human cirrhosis was not observed in all groups.

  As a result of the histopathological examination, in the group (Group 3-5) to which high fat diet + carbon tetrachloride was administered once, only accumulation of small lipid droplets was observed in the hepatocytes, but high fat diet + four In the group administered with carbon chloride 4 times or more, inflammatory cell infiltration around the central vein (inflammation), balloon-like enlargement of hepatocytes around the central vein, and fibers around the central vein reported in human NASH in clinical practice. Observed. Among the groups administered with the high fat diet + carbon tetrachloride four times or more, the high fat diet + carbon tetrachloride four times administration group (Group 7) and the high fat diet + carbon tetrachloride six times administration group (Group 8) 1 out of 5 cases, 3 cases out of 5 cases in the high-fat diet + carbon tetrachloride 8 administration group (Group 9) match NASH diagnostic criteria (Type 4 in Table 3) classified by Matteoni et al. It became clear.

(D) Blood biochemical properties (Group 1, 9)
When serum AST, ALT, free fatty acid, and neutral fat were measured, the high-fat diet + carbon tetrachloride 8 administration group (Group 9) was significantly higher than the normal diet group (Group 1) ( Table 6).
AST, ALT, free fatty acid, and neutral fat have been reported to be higher in human NASH patients than in healthy individuals, and similar results to human NASH were obtained.

From these results, NASH-like lesions are not induced in mice only by administration of a high-fat diet alone, but NASH-like lesions are also induced in the case of feeding a high-fat diet and single and double administration of carbon tetrachloride. It became clear that not. In addition, even after 72 hours from the single administration of carbon tetrachloride, the symptoms did not progress, and it became clear that NASH-like lesions were not induced.
On the other hand, it became clear that NASH-like lesions were induced by administration of carbon tetrachloride four or more times, and that NASH-like lesions were induced strongly and frequently by increasing the number of administrations of carbon tetrachloride. .
Moreover, in the carbon tetrachloride 8 times administration group, it became clear that the blood biochemical property showed the result similar to human NASH.
The generation mechanism of NASH is not fully clarified, and there is no report that NASH is induced by damaging the liver multiple times. From these results, NASH-like lesions can be induced by administering a hepatopathy agent at a dose lower than 1 ml / kg body weight / time, which is usually an amount that causes hepatopathy, and four times or more. It became clear. In view of the unclear generation mechanism of NASH, the reason why NASH-like lesions are induced by the above method is unclear.

Example 2
Except for the carbon tetrachloride dosage of 0.25 mL / kg body weight, the carbon tetrachloride was subcutaneously administered 8 times (2 times / week) in the same manner as in Example 1.
As a result, NASH model mice having the histopathological findings shown in Table 7 and the liver symptoms shown in FIGS. This mouse exhibited inflammation around the central vein, resulting in fibrosis, and large droplet fatty liver and hepatocyte balloon-like swelling.

Claims (22)

  A NASH model animal obtained by ingesting a high fat diet and administering a hepatic disorder agent four or more times.   The NASH model animal according to claim 1, wherein the high-fat diet contains 40 to 60% by mass of fat.   The NASH model animal according to claim 1 or 2, wherein the hepatic disorder is administered in an amount of 0.05 to 0.5 mL / kg body weight / time.   The NASH model animal according to any one of claims 1 to 3, wherein the hepatopathy agent is administered 4 to 8 times.   The NASH model animal according to any one of claims 1 to 4, wherein the hepatopathy agent is administered subcutaneously.   The NASH model animal according to any one of claims 1 to 5, wherein the intake period of the high fat diet is 2 weeks or more.   The NASH model animal according to any one of claims 1 to 6, wherein the hepatopathy agent is carbon tetrachloride.   The NASH model animal according to any one of claims 1 to 7, wherein the NASH model animal is a mouse.   The NASH model animal according to any one of claims 1 to 8, which is a large fatty liver, is inflamed around a central vein in a lobule of the liver, and hepatocytes are swollen in a balloon-like manner.   The NASH model animal according to any one of claims 1 to 9, wherein the liver tissue is fibrotic.   The NASH model animal according to any one of claims 1 to 10, wherein at least one selected from serum AST, ALT, free fatty acid, and neutral fat is higher than that of an animal fed with a normal feed.   The AST in serum is 200 U / L or more, ALT is 300 U / L or more, free fatty acid is 1000 μEq / L or more, and neutral fat is 20 mg / dL or more. The NASH model animal according to Item.   A method for producing a NASH model animal, characterized by ingesting a high fat diet and administering a hepatic disorder agent four or more times.   The method for producing a NASH model animal according to claim 13, wherein the high-fat diet contains 40 to 60% by mass of fat.   The method for producing a NASH model animal according to claim 13 or 14, wherein the dose of the hepatic disorder agent is 0.05 to 0.5 mL / kg body weight / time.   The method for producing a NASH model animal according to any one of claims 13 to 15, wherein the hepatopathy agent is administered 4 to 8 times.   The method for producing a NASH model animal according to any one of claims 13 to 16, wherein the hepatopathy agent is administered subcutaneously.   The method for producing a NASH model animal according to any one of claims 13 to 17, wherein the intake period of the high fat diet is 2 weeks or more.   The method for producing a NASH model animal according to any one of claims 13 to 18, wherein the hepatic disorder is carbon tetrachloride.   The method for producing a NASH model animal according to any one of claims 13 to 19, wherein the NASH model animal is a mouse.   A NASH prophylactic / therapeutic agent screening method, wherein the NASH model animal according to any one of claims 1 to 12 is used.   The NASH model animal of any one of Claims 1-12 is used, The evaluation method of the NASH preventive treatment effect of a test substance characterized by the above-mentioned.