UDC

576.38:577.215.3:616.36-004

DOI

10.37482/2687-1491-Z142

Authors

Elena I. Lebedeva* ORCID: https://orcid.org/0000-0003-1309-4248
Andrey S. Babenka** ORCID: https://orcid.org/0000-0002-5513-970X
Anatoliy T. Shchastniy* ORCID: https://orcid.org/0000-0003-2796-4240

*Vitebsk State Order of Peoples’ Friendship Medical University (Vitebsk, Republic of Belarus)
**Belarusian State Medical University (Minsk, Republic of Belarus)

Corresponding author: Elena Lebedeva,
address: prosp. Frunze 27, Vitebsk, 210009, Republic of Belarus;
e-mail: lebedeva.ya-elenale2013@yandex.ru

Abstract

The aim of this paper was to evaluate the potential of the mRNA level of the Nos2 gene as a marker of fibrogenesis in rats at different stages of thioacetamide-induced liver fibrosis and cirrhosis. Materials and methods. The experiment involved 117 mature male Wistar rats weighing 190–210 g. Liver fibrosis and cirrhosis were induced by a thioacetamide solution administered through a gastric catheter at a dose of 200 mg per 1 kg of body weight 2 times a week. The dynamics of the process was studied at 9 timepoints over the course of 17 weeks. Nos2 mRNA level in the liver was detected by means of real-time polymerase chain reaction. The stage of fibrosis was determined in histological sections stained using the Mallory method according to the Ishak semi-quantitative scale. Results. Throughout the experiment, Nos2 mRNA had practically no reaction to the thioacetamide-induced liver fibrogenesis. At the mild fibrosis stage (F1), an insignificant rise in the level of Nos2 mRNA was noted (within 5 %). Intensive development of fibrosis (F2–F4/F5) and an increase in the production of extracellular matrix components were accompanied by an increase in the level of Nos2 mRNA with a peak value exceeding the initial level (control group value) by the factor of 1.69 (р < 0.05). At the point of transition of fibrosis to cirrhosis (F5), a decrease in the level of mRNA of the target gene was observed, and at the stage of definite cirrhosis (F6), a subsequent drop below the initial level was detected. Thus, according to the data obtained, Nos2 mRNA has the greatest potential as a marker of liver fibrogenesis at the stage of advanced fibrosis, but cannot act as a marker at the early stages. Moreover, Nos2 mRNA level cannot be used as a marker when assessing the degree of cirrhosis and its development dynamics.

Keywords

rat liver, thioacetamide, markers of liver fibrogenesis, real-time polymerase chain reaction, Nos2 mRNA expression

References

1. Kashfi K. Nitric Oxide in Cancer and Beyond. Biochem. Pharmacol., 2020, vol. 176. Art. no. 114006. DOI: 10.1016/j.bcp.2020.114006
2. Ahmad N., Ansari M.Y., Haqqi T.M. Role of iNOS in Osteoarthritis: Pathological and Therapeutic Aspects. J. Cell. Physiol., 2020, vol. 235, no. 10, pp. 6366–6376. DOI: 10.1002/jcp.29607
3. Kashfi K., Kannikal J., Nath N. Macrophage Reprogramming and Cancer Therapeutics: Role of iNOS-Derived NO. Cells, 2021, vol. 10, no. 11. Art. no. 3194. DOI: 10.3390/cells10113194
4. Iwakiri Y. Nitric Oxide in Liver Fibrosis: The Role of Inducible Nitric Oxide Synthase. Clin. Mol. Hepatol., 2015, vol. 21, no. 4, pp. 319–325. DOI: 10.3350/cmh.2015.21.4.319
5. Becerril S., Rodríguez A., Catalán V., Ramírez B., Unamuno X., Gómez-Ambrosi J., Frühbeck G. iNOS Gene Ablation Prevents Liver Fibrosis in Leptin-Deficient ob/ob Mice. Genes (Basel), 2019, vol. 10, no. 3. Art. no. 184. DOI: 10.3390/genes10030184
6. Iwakiri Y., Kim M.Y. Nitric Oxide in Liver Diseases. Trends Pharmacol. Sci., 2015, vol. 36, no. 8, pp. 524–536. DOI: 10.1016/j.tips.2015.05.001
7. Atik E., Onlen Y., Savas L., Doran F. Inducible Nitric Oxide Synthase and Histopathological Correlation in Chronic Viral Hepatitis. Int. J. Infect. Dis., 2008, vol. 12, no. 1, pp. 12–15. DOI: 10.1016/j.ijid.2007.03.010
8. Wang W., Zhao C., Zhou J., Zhen Z., Wang Y., Shen C. Simvastatin Ameliorates Liver Fibrosis via Mediating Nitric Oxide Synthase in Rats with Non-Alcoholic Steatohepatitis-Related Liver Fibrosis. PLoS One, 2013, vol. 8, no. 10. Art. no. e76538. DOI: 10.1371/journal.pone.0076538
9. Anavi S., Eisenberg-Bord M., Hahn-Obercyger M., Genin O., Pines M., Tirosh O. The Role of iNOS in Cholesterol-Induced Liver Fibrosis. Lab. Invest., 2015, vol. 95, no. 8, pp. 914–924. DOI: 10.1038/labinvest.2015.67
10. Li D., Song Y., Wang Y., Guo Y., Zhang Z., Yang G., Wang G., Xu C. Nos2 Deficiency Enhances Carbon Tetrachloride-Induced Liver Injury in Aged Mice. Iran. J. Basic Med. Sci., 2020, vol. 23, no. 5, pp. 600–605. DOI: 10.22038/ijbms.2020.39528.9380
11. Sadek K.M., Saleh E.A., Nasr S.M. Molecular Hepatoprotective Effects of Lipoic Acid Against Carbon Tetrachloride-Induced Liver Fibrosis in Rats: Hepatoprotection at Molecular Level. Hum. Exp. Toxicol., 2018, vol. 37, no. 2, pp. 142–154. DOI: 10.1177/0960327117693066
12. Spitzer J.A., Zheng M., Kolls J.K., Vande Stouwe C.V., Spitzer J.J. Ethanol and LPS Modulate NFkappaB Activation, Inducible NO Synthase and COX-2 Gene Expression in Rat Liver Cells in vivo. Front. Biosci. (Landmark Ed.), 2002, pp. 7, no. 1, pp. 99–108. DOI: 10.2741/spitzer
13. Kikuchi H., Katsuramaki T., Kukita K., Taketani S., Meguro M., Nagayama M., Isobe M., Mizuguchi T., Hirata K. New Strategy for the Antifibrotic Therapy with Oral Administration of FR260330 (a Selective Inducible Nitric Oxide Synthase Inhibitor) in Rat Experimental Liver Cirrhosis. Wound Repair Regen., 2007, vol. 15, no. 6, pp. 881–888. DOI: 10.1111/j.1524-475X.2007.00308.x
14. Zhang J., Li Y., Liu Q., Li R., Pu S., Yang L., Feng Y., Ma L. SKLB023 as an iNOS Inhibitor Alleviated Liver Fibrosis by Inhibiting the TGF-beta/Smad Signaling Pathway. RSC Adv., 2018, vol. 8, no. 54, pp. 30919– 30924. DOI: 10.1039/c8ra04955f
15. Wen Y., Lambrecht J., Ju C., Tacke F. Hepatic Macrophages in Liver Homeostasis and Diseases-Diversity, Plasticity and Therapeutic Opportunities. Cell. Mol. Immunol., 2021, vol. 18, no. 1, pp. 45–56. DOI: 10.1038/s41423-020-00558-8
16. Everhart J.E., Wright E.C., Goodman Z.D., Dienstag J.L., Hoefs J.C., Kleiner D.E., Ghany M.G., Mills A.S., Nash S.R., Govindarajan S., Rogers T.E., Greenson J.K., Brunt E.M., Bonkovsky H.L., Morishima C., Litman H.J. Prognostic Value of Ishak Fibrosis Stage: Findings from the Hepatitis C Antiviral Long-Term Treatment Against Cirrhosis Trial. Hepatology, 2010, vol. 51, no. 2, pp. 585–594. DOI: 10.1002/hep.23315
17. Bustin S.A., Benes V., Garson J.A., Hellemans J., Huggett J., Kubista M., Mueller R., Nolan T., Pfaffl M.W., Shipley G.L., Vandesompele J., Wittwer C.T. The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments. Clin. Chem., 2009, vol. 55, no. 4, pp. 611–622. DOI: 10.1373/clinchem.2008.112797
18. Hernández R., Martínez-Lara E., Del Moral M.L., Blanco S., Cañuelo A., Siles E., Esteban F.J., Pedrosa J.A., Peinado M.A. Upregulation of Endothelial Nitric Oxide Synthase Maintains Nitric Oxide Production in the Cerebellum of Thioacetamide Cirrhotic Rats. Neuroscience, 2004, vol. 126, no. 4, pp. 879–887. DOI: 10.1016/j.neuroscience.2004.04.010
19. Wallace M.C., Hamesch K., Lunova M., Kim Y., Weiskirchen R., Strnad P., Friedman S.L. Standard Operating Procedures in Experimental Liver Research: Thioacetamide Model in Mice and Rats. Lab. Anim., 2015, vol. 49, suppl. 1, pp. 21–29. DOI: 10.1177/0023677215573040
20. Yanguas S.C., Cogliati B., Willebrords J., Maes M., Colle I., van den Bossche B., de Oliveira C.P.M.S., Andraus W., Alves V.A.F., Leclercq I., Vinken M. Experimental Models of Liver Fibrosis. Arch. Toxicol., 2016, vol. 90, no. 5, pp. 1025–1048. DOI: 10.1007/s00204-015-1543-4
21. Ravichandra A., Schwabe R.F. Mouse Models of Liver Fibrosis. Methods Mol. Biol., 2021, vol. 2299, pp. 339–356. DOI: 10.1007/978-1-0716-1382-5_23
22. Novo E., Bocca C., Foglia B., Protopapa F., Maggiora M., Parola M., Cannito S. Liver Fibrogenesis: Un Update on Established and Emerging Basic Concepts. Arch. Biochem. Biophys., 2020, vol. 689. Art. no. 108445. DOI: 10.1016/j.abb.2020.108445
23. Liu Y., Meyer C., Xu C., Weng H., Hellerbrand C., ten Dijke P., Dooley S. Animal Models of Chronic Liver Diseases. Am. J. Physiol. Gastrointest. Liver Physiol., 2013, vol. 304, no. 5, pp. G449–G468. DOI: 10.1152/ajpgi.00199.2012
24. Philipp D., Suhr L., Wahlers T., Choi Y.-H., Paunel-Görgülü A. Preconditioning of Bone Marrow-Derived Mesenchymal Stem Cells Highly Strengthens Their Potential to Promote IL-6-Dependent M2b Polarization. Stem Cell Res. Ther., 2018, vol. 9, no. 1. Art. no. 286. DOI: 10.1186/s13287-018-1039-2
25. Palumbo P., Miconi G., Cinque B., Lombardi F., La Torre C., Dehcordi S.R., Galzio R., Cimini A., Giordano A., Cifone M.G. NOS2 Expression in Glioma Cell Lines and Glioma Primary Cell Cultures: Correlation with Neurosphere Generation and SOX-2 Expression. Oncotarget, 2017, vol. 8, no. 15, pp. 25582–25598. DOI: 10.18632/oncotarget.16106