Investigation of molecular and cellular mechanisms of liver fibrosis in rats with post-toxic hepatosis of various etiologies and with the use of oxidized dextran

I n t r o d u c t i o n . The development of liver cirrhosis, regardless of etiology, is based on the process of fibrosis and structural alteration of the organ. Regulation of this process is associated with a high level of TGF-β expression and suppression of apoptosis in hepatocytes. Oxidized dextran (OD) has a high antifibrotic activity and is able to change the functional state of the phagocytic cell, thus preventing the development of fibrosis and stimulating reparative processes in organs with post-toxic hepatosis and liver cirrhosis.

A i m . To study the molecular and cellular mechanisms of the effect of OD on the expression of epithelial-mesenchymal transition (EMT)-associated proteins during fibrosis and the development of liver cirrhosis in rats with post-toxic hepatosis.

M a t e r i a l s a n d m e t h o d s . In the experiment, 30 male Wistar rats weighing 280–320 g were used. The animals were divided into 2 groups. In group 1 rats (hepatosis), the post-toxic hepatosis was modeled by administration of a solution of CCl4 and ethyl alcohol. In rats from group 2, the post-toxic hepatosis was modeled in the same way, and OD was administered. The numerical density (Nai) of liver nonparenchymal cells expressing TGF-β (Kupffer cells, endothelial cells, fibroblasts) was calculated. The expression of E-cadherin, vimentin, SNAIL + SLUG by fibroblasts and hepatocytes was evaluated.

R e s u l t s . The numerical density (Nai) of hepatocytes expressing vimentin prevailed in the liver of group 1 rats (hepatosis), compared with that of group 2 animals (hepatosis + OD) on the 30th and 60th days. In animals of the 1^st (hepatosis) group on the 60th day, a 3-fold lower numerical density of hepatocytes expressing E-cadherin was noted in comparison with that in rats treated with OD (group 2). In animals of the 1st (hepatosis) group on the 30th and 60th days, a 2-fold and 5-fold higher numerical density of hepatocytes expressing SNAIL + SLUG was noted in comparison with that in rats of the 2nd group. The numerical density of fibroblasts expressing vimentin prevailed in the liver of group 2 rats (hepatosis + OD), compared with that of group 1 animals (hepatosis) on day 30. In animals of the 1^st (hepatosis) group on the 60th day, a 6-fold higher numerical density of fibroblasts expressing E-cadherin was noted in comparison with that in rats treated with OD (group 2). In animals of the 1st (hepatosis) group on the 30^th and 60th days, the numerical density of fibroblasts expressing SNAIL + SLUG was 6 and 7 times higher than in rats treated with OD (group 2). In the liver of animals of the 2^nd group (hepatosis + OD), the numerical density of cells expressing TGF-β was lower in comparison with that of animals of the 1^st  group (hepatosis) by 2.5 times on the 30th day and 3.6 times on the 60th day of the experiment.

C o n c l u s i o n . In post-toxic hepatosis, the expression of TGF-β, SNAIL + SLUG and vimentin proteins increases in liver parenchymal and nonparenchymal cells, contributing to the acquisition of a mesenchymal immunophenotype by cells, which leads to increased profibrotic activity and the development of liver cirrhosis. The use of OD in post-toxic hepatosis reduces the expression of vimentin, TGF-β and EMT-associated proteins in liver parenchymal and nonparenchymal cells, which decreases the severity of fi broplastic processes and prevents the development of liver cirrhosis.

1. Nilsson E., Anderson H., Sargenti K. et al. Clinical course and mortality by etiology of liver cirrhosis in Sweden: a population based, long-term follow-up study of 1317 patients. Aliment. Pharmacol. Ther. 2019;49(11):1421-1430. DOI: 10.1111/apt.15255.

2. Ye F., Zhai M., Long J. et al. The burden of liver cirrhosis in mortality: Results from the global burden of disease study. Front. Public Health. 2022;10:1-13. DOI: 10.3389/fpubh.2022.909455.

3. Garbuzenko D.V. Current strategies for targeted therapy of liver fibrosis. Bulletin of Siberian Medicine. 2022;21(3):154-165. DOI: 10.20538/1682-0363-2022-3-154-165. (In Russ.)

4. Zhang C., Yang M., Ericsson A.C. Function of macrophages in disease: current understanding on molecular mechanisms. Front. Immunol. 2021;12(620510):1-12. DOI: 10.3389/fimmu.2021.620510.

5. Dvoryashina I.A., Velikorodnaya Yu.I., Terentev A.V., Zagrebin V.L. Type I epithelial-mesenchymal transition as an important biological process in embryogenesis. Journal of Volgograd State Medical University. 2021;2(78):37-45. DOI: 10.19163/1994-9480-2021-2(78)-37-45. (In Russ.)

6. Xu W., Wang N.R., Wang H.F. et al. Analysis of epithelial- mesenchymal transition markers in the histogenesis of hepatic progenitor cell in HBV-related liver diseases. Diagn. Pathol. 2016;11(1):136. DOI: 10.1186/s13000-016-0587-y.

7. Tennakoon A.H., Izawa T., Kuwamura M., Yamate J. Pathogenesis of type 2 epithelial to mesenchymal transition (EMT) in renal and hepatic fi brosis. J. Clin. Med. 2016;5(1):4. DOI: 10.3390/jcm5010004.

8. Karpov M.A., Klochin V.D., Nadeev A.P. et al. Structural changes in the liver in post-toxic cirrhosis and its treatment with oxidized dextran. Immunohistochemical research. Bulletin of Experimental Biology and Medicine. 2022;174(9):392-395. DOI: 10.47056/0365-9615-2022-174-9-392-395. (In Russ.)

9. Yang J., Antin P., Berx G. et al. Guidelines and definitions for research on epithelial-mesenchymal transition. Nat. Rev. Mol. Cell. Biol. 2020;21(6):341-352. DOI: 10.1038/s41580-020-0237-9.

10. Dongre A., Weinberg R.A. New insights into the mechanisms of epithelial-mesenchymal transition and implications for cancer. Nat. Rev. Mol. Cell. Biol. 2019;20(2):69-84. DOI: 10.1038/s41580-018-0080-4.

11. Yu K., Li Q., Shi G., Li N. Involvement of epithelial-mesenchymal transition in liver fibrosis. Saudi J. Gastroenterol. 2018;24(1):5-11. DOI: 10.4103/sjg.SJG_297_17.

12. Chen S., Morine Y., Tokuda K. et al. Cancer associated fibroblast induced M2 polarized macrophages promote hepatocellular carcinoma progression via the plasminogen activator inhibitor 1 pathway. Int. J. Oncol. 2021;59(2):59. DOI: 10.3892/ijo.2021.5239.

13. Sarbaeva N.N., Ponomareva J.V., Milyakova M.N. Macrophages: diversity of phenotypes and functions, interaction with foreign materials. Genes and Cells. 2016;11(1):9-17. (In Russ.)