Yonsei Med J.  2018 Nov;59(9):1064-1071. 10.3349/ymj.2018.59.9.1064.

S100A4 Gene is Crucial for Methionine-Choline-Deficient Diet-Induced Non-Alcoholic Fatty Liver Disease in Mice

• Zhang YH
• Ma DQ
• Ding DP
• Li J
• Chen LL
• Ao KJ
• Tian YY

• KMID: 2422491
• DOI: http://doi.org/10.3349/ymj.2018.59.9.1064

Abstract

PURPOSE
To explore the influence of S100 calcium binding protein A4 (S100A4) knockout (KO) on methionine-choline-deficient (MCD) diet-induced non-alcoholic fatty liver disease (NAFLD) in mice.
MATERIALS AND METHODS
S100A4 KO mice (n=20) and their wild-type (WT) counterparts (n=20) were randomly divided into KO/MCD, Ko/methionine-choline-sufficient (MCS), WT/MCD, and WT/MCS groups. After 8 weeks of feeding, blood lipid and liver function-related indexes were measured. HE, Oil Red O, and Masson stainings were used to observe the changes of liver histopathology. Additionally, expressions of S100A4 and proinflammatory and profibrogenic cytokines were detected by qRT-PCR and Western blot, while hepatocyte apoptosis was revealed by TUNEL staining.
RESULTS
Serum levels of aminotransferase, aspartate aminotransferase, triglyceride, and total cholesterol in mice were increased after 8-week MCD feeding, and hepatocytes performed varying balloon-like changes with increased inflammatory cell infiltration and collagen fibers; however, these effects were improved in mice of KO/MCD group. Meanwhile, total NAFLD activity scores and fibrosis were lower compared to WT+MCD group. Compared to WT/MCS group, S100A4 expression in liver tissue of WT/MCD group was enhanced. The expression of proinflammatory (TNF-α, IL-1β, IL-6) and profibrogenic cytokines (TGF-β1, COL1A1, α-SMA) in MCD-induced NAFLD mice were increased, as well as apoptotic index (AI). For MCD group, the expressions of proinflammatory and profibrogenic cytokines and AI in KO mice were lower than those of WT mice.
CONCLUSION
S100A4 was detected to be upregulated in NAFLD, while S100A4 KO alleviated liver fibrosis and inflammation, in addition to inhibiting hepatocyte apoptosis.

Keyword

S100 calcium-binding protein A4; non-alcoholic fatty liver disease; fibrosis; inflammation; apoptosis

MeSH Terms

Animals
Apoptosis
Aspartate Aminotransferases
Blotting, Western
Calcium
Carrier Proteins
Cholesterol
Collagen
Cytokines
Fibrosis
Hepatocytes
In Situ Nick-End Labeling
Inflammation
Liver
Liver Cirrhosis
Mice*
Non-alcoholic Fatty Liver Disease*
Triglycerides
Aspartate Aminotransferases
Calcium
Carrier Proteins
Cholesterol
Collagen
Cytokines

Figure

• 

  Fig. 1 S100A4 KO improves liver function and blood lipid-related parameters in MCD diet-induced non-alcoholic fatty liver disease mice. Influences of S100A4 KO on serum ALT (A), AST (B), TC (C), and TG (D) levels in mice. *p<0.05 compared to MCS group, †p<0.05 compared to WT/MCD group. S100A4, S100 calcium binding protein A4; MCD, methionine-choline-deficient; MCS, methionine-choline-sufficient; KO, knockout; WT, wild-type; ALT, aminotransferase; AST, aminotransferase; TG, triglyceride; TC, total cholesterol.

• 

  Fig. 2 Effect of S100A4 KO on liver morphology in MCD diet-induced non-alcoholic fatty liver disease mice. S100A4, S100 calcium binding protein A4; KO, knockout; WT, wild-type; MCD, methionine-choline-deficient; MCS, methionine-choline-sufficient.

• 

  Fig. 3 Relative S100A4 mRNA and protein expressions in liver tissues of mice detected by qRT-PCR (A) and Western blot (B and C). *p<0.05 compared to WT/MCS group. S100A4, S100 calcium binding protein A4; MCD, methionine-choline-deficient; MCS, methionine-choline-sufficient; KO, knockout; WT, wild-type.

• 

  Fig. 4 S100A4 KO reduces liver tissue damage and inflammatory cell infiltration in MCD diet-induced NAFLD mice. (A) HE staining, ORO staining, and Masson staining were used to observe pathological changes of liver tissues in each group (×400); (B–E) Comparison of steatosis (B), lobular inflammation (C), hepatocyte ballooning (D), total NAS (E), and fibrosis (F) in liver tissues of mice in each group. *p<0.05 compared to MCS group, †p<0.05 compared to WT/MCD group. S100A4, S100 calcium binding protein A4; MCD, methionine-choline-deficient; MCS, methionine-choline-sufficient; KO, knockout; WT, wild-type; HE, hematoxylin and eosin; ORO, Oil Red O; NAFLD, non-alcoholic fatty liver; NAS, NAFLD activity scores.

• 

  Fig. 5 S100A4 KO on expressions of proinflammatory cytokines (TNF-α, IL-1β, and IL-6) (A) and profibrogenic cytokines (TGF-β1, COL1A1 and α-SMA) (B) in liver tissues of MCD diet-induced non-alcoholic fatty liver disease mice detected by qRT-PCR. *p<0.05 compared to MCS group, †p<0.05 compared to WT/MCD group. S100A4, S100 calcium binding protein A4; MCD, methionine-choline-deficient; MCS, methionine-choline-sufficient; KO, knockout; WT, wild-type.

• 

  Fig. 6 S100A4 KO on hepatocyte apoptosis in MCD diet-induced non-alcoholic fatty liver disease mice evaluated by TUNEL staining (×400). Black arrows indicate hepatocyte apoptosis. *p<0.05 compared to MCS group, †p<0.05 compared to WT/MCD group. S100A4, S100 calcium binding protein A4; MCD, methionine-choline-deficient; MCS, methionine-choline-sufficient; KO, knockout; WT, wild-type.

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