J Korean Med Sci.  2021 Apr;36(14):e90. 10.3346/jkms.2021.36.e90.

Increased Expression of S100B and RAGE in a Mouse Model of Bile Duct Ligation-induced Liver Fibrosis

• Park JW
• Kim MJ
• Kim SE
• Kim HJ
• Jeon YC
• Shin HY
• Park SJ
• Jang MK
• Kim DJ
• Park CK
• Choi EK

• KMID: 2514901
• DOI: http://doi.org/10.3346/jkms.2021.36.e90

Abstract

Background
Liver fibrosis is defined as the accumulation of the extracellular matrix and scar formation. The receptor for advanced glycation end products (RAGE) has been demonstrated to participate in fibrogenesis. S100B is a ligand of RAGE and exerts extracellular functions by inducing a series of signal transduction cascades. However, the involvement of S100B and RAGE in cholestasis-induced liver fibrosis remains unclear. In this study, we investigated S100B and RAGE expression during liver fibrosis in mice that underwent common bile duct ligation (BDL).
Methods
BDL was performed in 10-week-old male C57BL/6J mice with sham control (n = 26) and BDL (n = 26) groups. Expression levels of S100B, RAGE and fibrotic markers in the livers from both groups at week 1 and 3 after BDL were examined by western blot and quantitative real-time reverse transcription polymerase chain reaction analysis. Liver fibrotic changes were examined by histological and ultrastructural analysis.
Results
Histological staining with Sirius Red and the evaluation of the messenger RNA expression of fibrotic markers showed noticeable periportal fibrosis and bile duct proliferation. S100B was mainly present in bile duct epithelial cells, and its expression was upregulated in proportion to the ductular reaction during fibrogenesis by BDL. RAGE expression was also increased, and interestingly, triple immunofluorescence staining and transmission electron microscopy showed that both S100B and RAGE were expressed in proliferating bile duct epithelial cells and activated hepatic stellate cells (HSCs) of the BDL livers. In addition, in rat HSCs (HSC-T6), treatment with recombinant S100B protein significantly increased fibrotic markers in a dose-dependent manner, and RAGE small interfering RNA (siRNA) suppressed S100B-stimulated upregulation of fibrotic markers compared with cells treated with scramble siRNA and S100B.
Conclusion
These findings suggest that the increased expression of S100B and RAGE and the interaction between S100B and RAGE may play an important role in ductular reaction and liver fibrosis induced by BDL.

Keyword

S100B; Receptor for Advanced Glycation End Products; Liver Fibrosis; Bile Duct Ligation

Figure

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  Fig. 1 Development of liver fibrosis after BDL. (A, B) Liver sections from sham control (upper panels) and BDL (lower panels) mice were examined by hematoxylin and eosin staining (A) and Sirius Red staining (B) at weeks 1 and 3 after BDL. (C) The mRNA levels of fibrotic marker genes at weeks 1 and 3 after BDL were determined by quantitative real-time reverse transcription polymerase chain reaction and normalized to β-actin (n = 9). The data represent the mean ± standard error of mean of three independent experiments. Scale bars, 25 μm.BDL = bile duct ligation, mRNA = messenger RNA, α-SMA = α-smooth muscle actin, TIMP-1 = tissue inhibitor of metalloproteinases-1, Col1a1 = collagen type I.*P < 0.05, **P < 0.01.

• 

  Fig. 2 Upregulated S100B expression in the livers of BDL mice. (A, B) Expression levels of S100B protein (A) and mRNA (B) in the livers from sham control and BDL mice was determined by Western blot analysis (A) and quantitative real-time reverse transcription polymerase chain reaction (B) and normalized to β-actin (n = 9) at each time point. The data represent the mean ± standard error of mean of three independent experiments. (C) Immunohistochemical staining of S100B in liver tissues from sham control and BDL mice. Note that an increased number of S100B-positive cells was found in the bile duct of BDL liver. Scale bars, 25 μm.BDL = bile duct ligation, mRNA = messenger RNA.**P < 0.01, ***P < 0.001.

• 

  Fig. 3 Subcellular localization of S100B and RAGE in the livers of BDL mice. (A) Expression of RAGE protein in the livers of sham control and BDL mice at 1 week and 3 weeks after operation was analyzed by Western blot analysis (left panel) and normalized to β-actin (right panel) (n = 9). The data represent the mean ± standard error of mean of three independent experiments. (B, C) Triple immunofluorescence staining of S100B, RAGE, and α-SMA (B) or CK19 (C) were performed in the liver sections of sham control and BDL mice at 3 weeks after operation. Note the colocalization of S100B and RAGE in α-SMA-positive activated HSCs (B) as well as CK19-positive bile duct epithelial cells (C) in the BDL livers. Green, S100B; red, α-SMA, and CK19; white, RAGE. Scale bars, 20 μm.RAGE = receptor for advanced glycation end products, BDL = bile duct ligation, α-SMA = α-smooth muscle actin, CK19 = cytokeratin19.*P < 0.05, **P < 0.01, ***P < 0.001.

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  Fig. 4 Double immunogold labeling of S100B and RAGE in the livers of BDL mice. The liver sections from sham control and BDL mice at week 3 were incubated with rabbit monoclonal anti-S100B (1:100) and mouse monoclonal anti-RAGE (1:100), followed by incubation with goat anti-rabbit IgG-conjugated 15 nm gold particles for S100B and goat anti-mouse IgG-conjugated 25 nm gold particles for RAGE. Note that immunogold particles indicating S100B (arrows) and RAGE (arrowhead) are present in the cytoplasm and microvilli of bile duct epithelial cells (upper panels), and in the HSCs, both particles were found in the nucleus and its environs (lower panels). Scale bars, 2 μm.RAGE = receptor for advanced glycation end products, BDL = bile duct ligation, HSC = hepatic stellate cell, IgG = immunoglobulin G, N = nuclei, M = mitochondria, L = lumen, LD = lipid droplet.

• 

  Fig. 5 S100B regulates HSC activation via RAGE. (A, B) HSC-T6 cells were treated with recombinant mouse S100B protein in a dose-dependent manner. After 12 hours, the cells were lysed, and HSC activation-related protein and gene expression levels were detected by Western blotting (A) and qRT-PCR (B), respectively. (C, D) HSC-T6 cells were transiently transfected with either SCR or si-RAGE in a dose-dependent manner, then incubated with 25 ng of recombinant S100B protein for 12 hours and analyzed by Western blotting (C) and qRT-PCR (D). The data represent the mean ± standard error of mean of three independent experiments.RAGE = receptor for advanced glycation end products, Col1a1 = collagen type I, α-SMA = α-smooth muscle actin, GAPDH = glyceraldehyde 3-phosphate dehydrogenase, mRNA = messenger RNA, PBS = phosphate-buffered saline, SCR = scrambled, si-RAGE = small interfering RNA-mediated knockdown of endogenous RAGE, qRT-PCR = quantitative real-time reverse transcription polymerase chain reaction, HSC = hepatic stellate cell.*P < 0.05, **P < 0.01, ***P < 0.001.

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