Cellular and Molecular Mechanisms of Toxic Liver Fibrosis in Rats Depending on the Stages of Its Development

Abstract

The aim is to study the cellular and molecular features of toxic liver fibrosis in rats and its dependence on development stages of this pathological condition.

Materials and methods: Liver fibrogenesis in male Wistar rats was induced with the thioacetamide solution by introducing into the stomach with a probe at a dose of 200 mg/kg of animal body weight 2 times per week. The process dynamics was studied at 5 time points (control, week 3, week 5, week 7, and week 9). The mRNA levels of tweak, fn14, ang, vegfa, cxcl12, and mmp-9 genes in liver were detected by real-time polymerase chain reaction. Immunohistochemical study was performed on paraffin sections. The CD31, CD34, CK19, α-SMA, FAP, CD68, CD206, CX3CR1, and CD45 cells were used as markers. Fibrosis degree was determined in histological sections, stained in line with the Mallory technique, according to the Ishak's semi-quantitative scale.

Results: Two simultaneously existing morphologically heterogeneous populations of myofibroblasts expressing different types of markers (FAP, α-SMA) were identified in rat liver. Prior to the onset of transformation of fibrosis into cirrhosis (F1-F4, weeks 3-7), FAP⁺ and SMA⁺ cells were localized in different places on histological specimens. All stages of liver fibrosis development were accompanied by an increase in the number (p=0.0000), a change in the phenotypic structure and functional properties of macrophages. The CK19⁺ cells of the portal areas differentiated into cholangiocytes that formed interlobular bile ducts and ductules, as well as hepatocytes that formed rudiments of new hepatic microlobules. Pathological venous angiogenesis and heterogeneity of endotheliocytes of the intrahepatic vascular bed were detected. Two options for changes in mRNA expression of the selected genes were identified. The level of the fn14 and mmp-9 mRNAs at all stages of fibrosis was higher (p=0.0000) than in control rats. For tweak, ang, vegfa, and cxcl12 mRNAs, the situation was the opposite - the level of genes decreased (p=0.0000). There were strong and moderate correlations between the studied target genes (p<0.05).

Conclusion: It was established that the stages of toxic fibrosis had morphological and molecular genetic features. The FAP⁺ cells make the main contribution to development of portal and initial stage of bridging fibrosis. The stellate macrophages and infiltrating monocytes/ macrophages can potentially be used for development of new therapeutic strategies for liver pathology treatment. One should take into account the features of the markers' expression by endothelial cells during the study of the intrahepatic vascular bed. Joint study of genes is a necessary ad-hoc parameter in fundamental and preclinical research.

Keywords: Ishak’s scale; immunohistochemistry; mRNA expression; rat liver fibrogenesis; toxic fibrosis.

Figure 1.. Pieces of rat liver 3 weeks (a, b), 7 weeks (c), and 9 weeks (d) after the start of the experiment:

staining with hematoxylin and eosin, ×60 (a); staining in line with the Mallory technique, ×40 (b, c), ×20 (d); (a) perinuclear edema (arrows); (b) fibrous connective tissue in the portal area (arrows); (c) fibrous septa between portal areas (arrows); (d) false hepatic lobule (oval frame), angiogenesis in the portal areas (arrows)

Figure 2.. Pieces of rat liver of the control group (b), 5 weeks (a), and 3 weeks (c, d) after the start of the experiment:

staining in line with the Mallory technique, ×20 (a); immunohistochemical staining for CD31, additional staining with Mayer’s hematoxylin, ×40 (b), ×20 (d); immunohistochemical staining for CD34, additional staining with Mayer’s hematoxylin, ×20 (c); (a) irregularly shaped interlobular vein (arrow); (b) CD31⁺ cells in the sinusoids of the central area of the classical hepatic lobule (round frame); (c) CD34⁺ cells in interlobular veins (arrows); (d) CD31⁺ cells in sinusoids (arrows) and interlobular vein (star)

Figure 3.. Change in the area of CD31⁺, CD34⁺, CK19⁺ cells with the development of fibrous connective tissue

Figure 4.. Pieces of the rat liver of the control group (a, b) and 9 weeks (c, d) after the start of the experiment:

immunohistochemical staining for FAP, additional staining with Mayer’s hematoxylin, ×40 (a, c); immunohistochemical staining for α-SMA, staining with Mayer’s hematoxylin, ×40 (b, d); (a) FAP⁺ cells are missing; (b) α-SMA⁺ cells are missing; (c) FAP⁺ cells in fibrous septa (arrows); (d) α-SMA⁺ cells in sinusoids (arrows)

Figure 5.. Change in the number of CD68⁺, CD206⁺, CX3CR1⁺, CD45⁺, FAP⁺, and α-SMA⁺ cells with the fibrous connective tissue growth

Figure 6.. Pieces of the rat liver of the control group (a, b, c), 5 weeks (d) and 7 weeks (e, f) after the start of the experiment:

immunohistochemical staining for CD68, staining with Mayer’s hematoxylin, ×40 (a), ×60 (d); immunohistochemical staining for CD206, additional staining with Mayer’s hematoxylin, ×40 (b); ×60 (e); immunohistochemical staining for CX3CR1, additional staining with Mayer’s hematoxylin, ×40 (c), ×60 (f); (a, d) CD68⁺ cells in sinusoids (arrows); (b) CD206⁺ cells are missing; (c) CX3CR1⁺ cells are missing; (e) CD206⁺ cells in sinusoids (arrows); (f) a group of CX3CR1⁺ cells in the portal area (oval frame)

Figure 7.. Pieces of the rat liver of the control group (a), 3 weeks (b), and 5 weeks (c, d) after the start of the experiment:

immunohistochemical staining for CK19, staining with Mayer’s hematoxylin, ×40 (a), ×20 (b, c), ×100 (d); (a, b, c) interlobular bile ducts (arrows); (d) accumulation of CK19⁺ cells without a lumen (arrow), accumulation of CK19⁺ cells with a developing lumen (round frame), accumulation of CK19⁺ cells with a formed lumen (star)

Figure 8.. Changes in the mRNA expression level of the tweak, fn14, ang, vegfa, cxcl12, and mmp-9 genes during the fibrous connective tissue growth

Figure 9.. Correlations between the tweak, fn14, ang, vegfa, cxcl12, and mmp-9 genes during the fibrous connective tissue growth

Figure 10.. Percentage of the absolute number of mRNA copies of the tweak, fn14, ang, vegfa, cxcl12, and mmp-9 genes during the fibrous connective tissue growth