The dynamics of microRNAs level associated with pathological venous angiogenesis in experimental toxic liver fibrosis in rats

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Abstract

BACKGROUND: It is known that miRNAs are important in liver fibrogenesis. However, their use as targets for early diagnosis and treatment of fibrosis is far from use in clinical practice. Angiogenesis and sinusoid capillarization are important histological features of the process. Studies regarding the role of miRNAs in pathological angiogenesis and sinusoid capillarization are insufficient.

AIM: To study the molecular targets (miRNAs and mRNAs) dynamics of associated with pathological angiogenesis in toxic fibrosis of the liver; to evaluate the relationship of the selected molecular factors to the processes of restructuring the intrahepatic vascular system.

METHODS: Fibrosis and subsequent cirrhosis of the liver in rats of the Wistar line (males) were induced for 17 weeks by a freshly prepared solution of thioacetamide. The level of miRNA-19а-3р, miRNA-29b-3р, miRNA-29b-1-5p, miRNA-34b-5р, miRNA-125b-5р, miRNA-130a-5p, miRNA-195-5р, miRNA-449а-5р, miRNA-449с-5р, miRNA-466d, miRNA-489-3р, miRNA-495, miRNA-664-3р, miRNA-3085, miRNA-3558-3р in fresh frozen liver samples, was determined by Two-tailed RT-qPCR.

RESULTS: In this study, we found that throughout the experiment, the relative level of microRNAs varied in a wide range of values (10–3–104 rel. units). In most cases, it decreased at the point of transition from fibrosis to cirrhosis, while growth was observed only for microRNA-29b-3p. Statistically significant correlation relationships were established between microRNAs and the number of interlobular veins, interlobular arteries, sinusoids, and the area of connective tissue (p <0.05).

CONCLUSION: A joint analysis of morphological and molecular-genetic parameters allowed us to suggest that within the framework of the current experimental model of liver fibrosis and cirrhosis, the restructuring of the intrahepatic vascular bed and the progression of fibrosis are associated with the dynamics of the level of a number of microRNAs that we studied and Ang mRNA level.

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About the authors

Elena I. Lebedeva

Vitebsk State Order of Peoples’ Friendship Medical University

Author for correspondence.
Email: lebedeva.ya-elenale2013@yandex.ru
ORCID iD: 0000-0003-1309-4248
SPIN-code: 4049-3213

PhD, Cand. Sci. (Biology), Associate Professor

Belarus, Vitebsk

Andrei S. Babenka

Belarusian State Medical University

Email: labmdbt@gmail.com
ORCID iD: 0000-0002-5513-970X
SPIN-code: 9715-4070

PhD, Cand. Sci. (Chemistry), Associate Professor

Belarus, Minsk

Anatoly T. Shchastniy

Vitebsk State Order of Peoples’ Friendship Medical University

Email: rectorvsmu@gmail.com
ORCID iD: 0000-0003-2796-4240
SPIN-code: 3289-6156

PhD, Cand. Sci. (Biology), Associate Professor

Belarus, Vitebsk

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Supplementary files

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1. JATS XML
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2. Table 1. List of microRNAs included in the study, target microRNAs

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3. Fig. 1. Histological preparations of rat liver, microphoto: control group (a), after 3 (b), 5 (c), 7 (d), 9 (e), 11 (f), 13 (g), 15 (h, i), 17 (j) weeks after the start of the experiment. Staining using the Mallory method. Mag.: ×100 (g, h, j); ×200 (d, i); ×400 (a, b, c, e, f); a — central vein (arrow); b — incomplete fibrous connective tissue septa (arrow); c — fibrous connective tissue septa (arrows); d — pericellular fibrosis (arrows); e — false hepatic lobule (oval frame); f — thick fibrous connective tissue septa (arrows); g, h — false hepatic lobules of different diameters and shapes, pronounced pathological angiogenesis (arrows); and i — bile ducts (arrows), ductal reaction (oval frame); j — pronounced destruction of the parenchyma.

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4. Fig. 2. Histological preparations of rat liver, microphoto: control group (а), in 3 (b), 9 (c), 11 (d), 13 (g), 15 (e, h), (f) weeks after the start of the experiment. Immunohistochemical staining for CK19, counterstaining with Mayer’s hematoxylin. Staining using the Mallory method (h). Mag.: ×400 (a, c, e, g, h), ×200 (b, d, f); a–e — CK19+ cells (arrows), f — bile ducts (arrows), g — process of formation of hepatic microlobules from CK19+ cells (oval frame), h — new hepatic microlobules (oval frames).

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5. Fig. 3. Histological preparations of rat liver, microphotographs: control group (a), 3 (b), 5 (c), 11 (d), 13 (e), 17 (f) weeks after the start of the experiment. Immunohistochemical staining for CD31, counterstaining with Mayer’s hematoxylin. Mag.: ×400 (а, c, d, e), ×200 (b, f); а — CD31+ cells in the sinusoidal capillaries of the central zone of the classical hepatic lobule (oval frame); b–f — CD31+ cells in the sinusoidal capillaries (arrows) and in the blood vessels of the portal zones and fibrous septa (stars).

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6. Fig. 4. Changes in the area of CD31+ cells at different stages of the study.

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7. Fig. 5. Change in the area of CK19+ cells at different stages of the study.

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8. Fig. 6. Dynamics of the relative level of microRNAs during fibrogenesis with transition to cirrhosis.

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9. Fig. 7. Changes in morphological, immunohistochemical and molecular genetic parameters, likely associated with the processes of initiation and progression of fibrosis with transition to cirrhosis.

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10. Fig. 8. Correlation between the level of microRNAs, the number of interlobular veins, interlobular arteries, sinusoids, connective tissue area and CD31+, CK19+ cells.

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