Development of Non-Alcoholic Steatohepatitis (NASH) Model
Using a Chimeric Mouse with Humanized Livers (PXB-Mouse®)and Evaluation of Efficacy of Pioglitazone○ Ibrohimjon Shukurov, Naomi Hikawa, Shiruku Hosoi, Nahoko Matsuki,

Kousuke Morizumi, Seiichi Katayama, Naoyuki Hironaka, Katsuhide Nishi

Objective

Results
Body Weight25013274256084

Plasma ALT**

013µg/g Liver

#**

**

NormalNASH
NASH+Pioglitazone

270

**
**

4Day after administration

300

**

6

NormalNASH
NASH+Pioglitazone

5#8

mg/mL

10274256**70600
400
2000

Plasma T-CHO

0013274256300

Day after administration**#2100084(Sirius red staining)

4200

70

Fibrosis area

6400

NormalNASH
NASH+Pioglitazone

NASH +Pioglitazone%U/L100

NASH

8500#Normal

10*

600

#

#800

84

Day after administration

700

##**

1000

10******* **15(µg/g Liver)

1200

12200

現在、非アルコール性脂肪肝炎（NASH）の治療薬がなく，さらにヒトのNASH病態の複雑さを模倣する適切な動物モデルがない．そこで，私達はヒト肝キメラマウス（ PXB-Mouse®）を用いてNASHモデルを作製し，ピオグリタゾン（PTZ ）の作用を検討した．PXB-Mouse®に独自に開発したコリン不含・L-アミノ酸規定（CDAA ）飼料を84日間自由摂取させることでNASH モデルを作製した．給餌 期間中，PTZ を1 日 1 回 経口投与した．NASH モデル群では血漿ALT, T-Cho量 , 血清サイトカインおよび肝臓中のヒドロキシプロリン（HYP）量が増加し，病理組織学的検査で肝臓の線維化が認められた．一方，PTZ 投与群では血漿 ALT, T-Cho量 ,血清サイトカインおよび肝臓中のHYP量および線維化面積が減少したことから, PTZはNASHの病態を改善すると考えられた．

Liver HYP

h-Alb14200Liver Parameters

mg/mL

Summary in Japanese

Body Weight & Plasma ParametersgNon-alcoholic steatohepatitis (NASH) is characterized by accumulationof fat (steatosis), inflammation, and fibrosis in the liver without alcohol
consumption. It is serious threat to health. However, currently, there are
no approved therapeutic agents for NASH, and it is considered there isno animal model that appropriately represents the complexity ofh u m a n N AS H d i s e a s e . Ch i m e r i c m i c e w i t h h u m a n ize d l i ve r s(PXB-Mouse®) could show potential for developing new drugs for humanliver diseases.
There are research reports which highlights potential benefits of
Pioglitazone (PTZ) in NASH.
The aim of this study is to develop a NASH model using PXB-Mouse®,which could be promising tool for investigating therapeutic agents for
NASH, and evaluate the potential therapeutic benefits of PTZ.

NormalNASH

0NASH+
Pioglitazone

Normal

NASHNASH+
Pioglitazone*: P<0.05, **:P<0.01; Normal group vs NASH group (Student’s t-test)#: P<0.05, ##:P<0.01; NASH group vs NASH+Pioglitazone group (Student’s t-test)
Each value represents the mean±S.E. (N=3-5)

HEMaterials and Methods● AnimalPXB-mouse® (PhoenixBio Co., Ltd.), ♂, 18-22 weeks old● DietCRF-1:AS:
CDAA:

Oriental Yeast Co., Ltd.
Oriental Yeast Co., Ltd.
The choline-deficient L-amino acid-defined 50kcal% fat
containing diet (patented by Mediford Corporation, Patent
no.: JP6211035B2).

● DrugPioglitazone HCl:
FUJIFILM Wako Pure Chemical Corporation,
PPAR-γ agonist.

● Microscopic Examination (HE stain)

LobularSteatosis Inflammation

Group(3)Normal(5)NASH
NASH+Pioglitazone (4)2.3 ± 0.3
3.0 ± 0.0
2.8 ± 0.3

0.7 ± 0.3
0.6 ± 0.2
0.5 ± 0.3

Ballooning
hepatocyte
0.0 ± 0.0
1.0 ± 0.0
1.0 ± 0.0● Measurement parameters
Serum Human & Mouse Cytokine and Chemokine:
Bio-Plex Pro Human Cytokine 27-Plex Panel, Mouse
Cytokine 23-Plex Panel (Bio-Rad Laboratories, Inc.),
at terminal point.
Plasma ALT, T-Cho:
DRI-CHEM FDC7000iZ (Fujifilm Chemical Co., Ltd.), ALT
measurement was performed per two weeks, T-Cho
measurement was performed at terminal point.
Blood h-Alb: Biochemistry Automatic Analyzer (7180 Clinical Analyzer,Hitachi High-tech Corporation), per two weeks.
Liver Hydroxyproline (HYP):
Measurement was performed at terminal point.
Histopathological observation:Hematoxylin and Eosin stain (HE), Sirius Red stain (SR),Anti-human Lamin stain (Lamin)
Test group

Diet

Normal
CRF-1, ASNASHCDAA
NASH+Pioglitazone CDAA

Administration
0.5 w/v% MC
0.5 w/v% MC
Pioglitazone (PTZ)

Frequency& Period

Dose(mg/kg)

Ｎ

QD for84 days0020

354

hepatocytes with large sized lipid droplets
Ballooning hepatocytes and Mallory–Denk-body-likeaggregates
inflammatory cell
central vein
Grade sign: 0, none; 1, minimal; 2, mild; 3, moderate; 4, marked.
Each value represents the mean±S.E. of the histopathology score.
Figures in parentheses are number of animals with tissues examinedhistopathologically.

SR▲ fibrosis

Blood collection
TerminalLamin
Day 0132742567084

Administration (p.o.)Diet feeding

Conclusion

PTZ’s action mechanism in the NASH is considered as follows. PTZ
activates PPAR-γ in the adipose tissue, and it decreases theinsulin resistance. In addition, it regulates free fatty acid (FFA)
release and triglyceride (TG) circulation. As the result of FFA
regulation, the FFA and lipid droplets levels decrease in the liver.
From these changes, the liver cells are exposed to less stress ordamage, which leads to decrease in attraction of monocyte, NKcells etc., and lower level of proinflammatory markers like MCP-1,IL-6, IL-8, IP-10. Resultantly, PTZ contribute to a reduction ofinflammation within the liver.
As a summary, NASH model mice using PXB-Mouse® could be
helpful for new drug development.

Mouse Cytokine

Human Cytokine
450

**5ND
Normal

NASH

NASH+
Pioglitazone

30250

pg/mL

pg/mL

40

1040300
#200

20150

20100

1000050

Normal

NASH

NASH+
PioglitazoneMCP-1 (CCL2)

50350

6015IP-10 (CXCL10)40080

200IL-8100pg/ml

IL-625pg/mL

Pioglitazone (PTZ) decreased the lipid accumulation in the liver,reflected by lower T-Cho level, as well as lower levels of IL-8, IP-10,
MCP-1 and IL-6, and lower hepatic fibrosis area.

Positive reaction in nuclei of human cells.

NormalNASH

NASH+
Pioglitazone

Normal

NASH

NASH+
Pioglitazone**:P<0.01; Normal group vs NASH group (Student’s t-test)#: P<0.05; NASH group vs NASH+Pioglitazone group (Student’s t-test)
ND: Not detected
Each value represents the mean±S.E. (N=3-5)

Liver

In the present study, the NASH model mice (PXB-Mouse®)
showed a variety of parameter changes that resemble humanpathological conditions, such as elevated ALT, T-Cho, liver HYP,
proinflammatory cytokines and chemokines. In addition, theenhanced hepatic fibrosis was confirmed by SR staining in NASH
model animals. Higher steatosis and ballooning hepatocyte
grade were also confirmed. These findings suggest that the NASH
model mice using PXB-Mouse® could develop a human NASH-likepathology better than the previous animal models.

↓ IL-6↓ MCP-1↓ IP-10↓ IL-8↓ NK cells↓ Monocytes↓ T cells↓ FFA↑PPAR-γ activation↓Inflammation↓ Fibrosis

Adipose tissue

Pioglitazone↓ Insulin Resistance↓ FFA release↓ Triglyceride & Cholesterol circulation

Fabio Marra & Frank Tacke, Gastroenterology, 2014