Anti-apoptosis mechanism of the hepatoprotective effect of pyrimidine derivatives in in vivo studies
Institutes and Faculties
Main page \ Research and Innovations \ Publications \ Publishing activities of KFU \ Uchenye Zapiski Kazanskogo Universiteta \ Uchenye Zapiski Kazanskogo Universiteta. Seriya Estestvennye Nauki \ Archive

Anti-apoptosis mechanism of the hepatoprotective effect of pyrimidine derivatives in in vivo studies

A.A. Parfenov a*, A.B. Vyshtakalyuk a,b**, I.V. Galyametdinova a***, V.E. Semenov a,c****, V.V. Zobov a,b*****

aArbuzov Institute of Organic and Physical Chemistry, FRC Kazan Scientific Center, Russian Academy of Sciences, Kazan, 420088 Russia

bKazan Federal University, Kazan, 420008 Russia

cKazan National Research Technological University, Kazan, 420015 Russia

E-mail: *aimt66@gmail.com, **alex.vysh@mail.ru, ***iragal2009@yahoo.com, ****sve@iopc.ru, *****vz30608@mail.ru

Received February 28, 2022

 

ORIGINAL ARTICLE

Full text PDF

DOI: 10.26907/2542-064X.2022.2.231-248

For citation: Parfenov A.A., Vyshtakalyuk A.B., Galyametdinova I.V., Semenov V.E., Zobov V.V. Anti-apoptosis mechanism of the hepatoprotective effect of pyrimidine derivatives in in vivo studies. Uchenye Zapiski Kazanskogo Universiteta. Seriya Estestvennye Nauki, 2022, vol. 164, no. 2, pp. 231–248. doi: 10.26907/2542-064X.2022.2.231-248. (In Russian)

Abstract

This study investigates the effect of pyrimidine derivatives (Xymedon and its conjugate with L-ascorbic acid, both exhibiting hepatoprotective activity) on the apoptosis of rat liver cells against the background of exposure to carbon tetrachloride as a hepatotoxic agent. The characteristic markers of apoptosis affected by the considered compounds were identified. Modern multiplex analysis of the early apoptosis markers Akt, BAD, BCL-2, p53, Active Caspase-8, and Active Caspase-9 in rat liver homogenates using the MagPix system (MerkMillipore, USA) was selected as the main research method. The results obtained show that Xymedon and its derivative with L-ascorbic acid exert an anti-apoptotic effect by causing a significant decrease in the number of the early apoptosis markers BAD and Active Caspase-9. The derivative with L-ascorbic acid was also found to reduce p53 expression. Moreover, it was revealed that the studied pyrimidine derivatives normalize the biochemical markers of liver damage (transaminases, alkaline phosphatase, bilirubin, total protein, and albumin) and increase the level of glucose in the blood serum of rats against the background of acute toxic damage to the liver by carbon tetrachloride. Our findings are of the utmost theoretical and practical relevance for better understanding the mechanisms of the action of pyrimidine derivatives and for developing a highly effective hepatoprotective drug.

Keywords: pyrimidine derivatives, Xymedon, apoptosis, carbon tetrachloride, toxic liver damage

Acknowledgments. This study was funded by the Russian Foundation for Basic Research (project no. 20-315-90039). The work by A. Vyshtakalyuk, I. Galyametdimova, V. Semenov, and V. Zobov was supported in part by the state assignment to the FRC Kazan Scientific Center of the Russian Academy of Sciences.

Figure Captions

Fig. 1. Structural formula of the studied compounds: 1 – Xymedon; 2 – conjugate of Xymedon with     L-ascorbic acid.

Fig. 2. Microphotographs of the rat liver sections after a three-day exposure to CCl4: a – hematoxylin and eosin staining (1 – hydropic degeneration, 2 – Councilman body, 3 – macrovesicular adipose degeneration, 4 – balloon degeneration, 5 – microvesicular adipose degeneration); b) Sudan IV staining (orange drops are lipids).

Fig. 3. Effect of compounds 1 and 2 on the markers of cell cytolysis: a) aspartate aminotransferase (AST) activity; b) alanine aminotransferase (ALT) activity. 1 – differences from the intact group; 2 – differences from the control group on the same day. Levels of significance p determined using Tukey’s HDS and Games-Howell tests.

Fig. 4. Effect of compounds 1 and 2 on the bile and bilirubin excretion markers: a) alkaline phosphatase (AP) activity; b) concentration of total bilirubin; c) concentration of unconjugated bilirubin; d) concentration of conjugated bilirubin. 1 – differences from the intact group; 2 – differences from the control group on the same day; 3 – differences between the control group on day 4 and the control group on day 10. Levels of significance p determined using Tukey’s HDS and Games-Howell tests.

Fig. 5. Effect of compounds 1 and 2 on the markers of protein metabolism and glucose: a) albumin concentration; b) total protein concentration; c) glucose concentration. 1 – differences from the intact group; 2 – differences from the control group on the same day. Levels of significance p determined using Tukey’s HDS and Games-Howell tests.

Fig. 6. The effect of compounds 1 and 2 on the mean fluorescence intensity (MFI) of the markers of early apoptosis: a) MFI BAD; b) MFI BCL-2; c) MFI Akt. 1 – differences from the intact group; 2 – differences from the control group on the same day. Levels of significance p determined using Mann-Whitney test; critical level of significance р* = 0.002.

Fig. 7. Effect of compounds 1 and 2 on the mean fluorescence intensity (MFI) of activated caspases 9 and 8: a) MFI-activated caspase 9 (CASP9); b) MFI-activated caspase 8 (CASP8). 1 – differences from the intact group, 2 – differences from the control group on the same day. Levels of significance p determined using Mann-Whitney test; critical level of significance р* = 0.002.

Fig. 8. Effect of compounds 1 and 2 on the mean fluorescence intensity (MFI) of p53. 1 – differences from the intact group, 2 – differences from the control group on the same day. Levels of significance p determined using Mann-Whitney test; critical level of significance р* = 0.002.

References

  1. Arias I.M., Alter H.J., Boyer J.L., Cohen D.E., Shafritz D.A. The Liver: Biology and Pathobiology. Oxford, John Wiley & Sons, 2020. 1144 p.
  2. Izmailov S.G., Parshikov V.V. Izmailov S.G., Parshikov V.V. Xymedon: Mapping its present and future. Nizhegorod. Med. Zh., 2002, no. 3, pp. 81–87. (In Russian)
  3. Porfiriev A., Sharafutdinova D., Belyaev A., Parfenov A., Shubina V., Golubev A., Vyshtakaliuk A., Zobov V., Semenov V. The influence of the Xymedon conjugate with L-methionine on the regeneration of Schmidtea mediterranea planarians. BioNanoScience, 2020, vol. 10, no. 2, pp. 397–402. doi: 10.1007/s12668-020-00735-z.
  4. Vyshtakalyuk A.B., Semenov V.E., Zobov V.V., Galyametdinova I.V., Gumarova L.F., Parfenov A.A., Nazarov N.G., Lenina O.A., Kondrashova S.A., Latypov Sh. K., Cherepnev G.V., Shahyn M.S., Reznic V.S. Synthesis and primary evaluation of the hepatoprotective properties of novel pyrimidine derivatives. Russ. J. Bioorg. Chem., 2017, vol. 43, no. 5, pp. 604–611. doi: 10.1134/S106816201704015X.
  5. Vyshtakalyuk A., Parfenov A., Gumarova L., Nazarov N., Zobov V., Galyametdinova I., Semenov V. Comparative evaluation of hepatoprotective activity of Xymedon preparation derivatives with ascorbic acid and methionine. BioNanoScience, 2017, vol. 7, no. 4, pp. 616–622. doi: 10.1007/s12668-017-0461-8.
  6. Vyshtakalyuk A.B., Parfenov A.A., Gumarova L.F., Khasanshina L.R., Belyaev G.P., Nazarov N.G., Kondrashina D.A., Galyametdinova I.V., Semenov V.E., Zobov V.V. Conjugate of pyrimidine derivative, the drug xymedon with succinic acid protects liver cells. J. Biochem. Mol. Toxicol., 2020, vol. 35, no. 3, art. e22660, pp. 1–12. doi: 10.1002/jbt.22660.
  7. Vyshtakalyuk A.B., Parfenov A.A., Nazarov N.G., Gumarova L.F., Cherepnev G.V., Galyametdinova I.V., Zobov V.V., Semenov V.E. Hepato-, nephro-and pancreatoprotective effect of derivatives of drug Xymedon with biogenic acids under toxic influence of carbon tetrachloride in rats. BioNanoScience, 2018, vol. 8, no. 3, pp. 845–858. doi: 10.1007/s12668-018-0526-3.
  8. Vyshtakalyuk A.B., Semenov V.E., Sudakov I.A., Bushmeleva K.N., Gumarova L.F., Parfenov A.A., Nazarov N.G., Galyametdinova I.V., Zobov V.V. Xymedon conjugate with biogenic acids. Antioxidant properties of a conjugate of Xymedon with L-ascorbic acid. Russ. Chem. Bull., 2018, vol. 67, no. 4, pp. 705–711. doi: 10.1007/s11172-018-2126-3.
  9. Parfenov A.A., Vyshtakalyuk A.B., Gumarova L.F., Khasanshina L.R., Belyaev G.P., Nazarov N.G., Kondrashina D.A., Galyametdinova I.V., Zobov V.V., Semenov V.E. Xymedone conjugate with para-aminobenzoic acid. Estimation of hepatoprotective properties. Russ. Chem. Bull., 2019, vol. 68, no. 12, pp. 2307–2315. doi: 10.1007/s11172-019-2704-z.
  10. Vyshtakalyuk A.B., Nazarov N.G., Semenov V.E., Galyametdinova I.V., Diabankana R.G.K., Porfiriev A.G., Zobov V.V. Recovery of liver damaged by CCl4 under treatment by conjugate of drug Xymedon with L-ascorbic acid. Int. J. Pharm. Sci. Res., 2018, vol. 9, no. 10, pp. 4117–4126. doi: 10.13040/IJPSR.0975-8232.9(10).4117-26.
  11. Reznik V.S., Pashkurov N.G. Reactions of pyrimidinols and pyrimidinethiols with 2-chloroethanol and with 2-chloro-1-propanol. Bull. Acad. Sci. USSR, Div. Chem. Sci., 1966, vol. 15, no. 9, pp. 1554–1557. doi: 10.1007/BF00848915.
  12. Mironov A.N. (Ed.) Rukovodstvo po provedeniyu doklinicheskikh issledovanii lekarstvennykh sredstv [A Guide to Preclinical Drug Research]. Moscow, Grif i K, 2012. 536 p. (In Russian)
  13. Grjibovski A.M. Analysis of quantitative data in three or more independent groups. Ekol. Chel., 2008, no. 3, pp. 50–58 (In Russian)
  14. Slabnov Y.D., Cherepnev G.V., Karimova F.G., Garaev R.S. Effect of pyrimidine derivatives on adenylate cyclase system of immunocompetent cell regulation in vitro. Bull. Exp. Biol. Med., 1998, vol. 125, no. 6, pp. 588–590. doi: 10.1007/BF02445248.
  15. Cooper D.M., Cooper D.M.F. Regulation and organization of adenylyl cyclases and cAMP. Biochem. J., 2003, vol. 375, no. 3, pp. 517–529. doi: 10.1042/BJ20031061.
  16. Wahler G.M., Sperelakis N. Regulation of cardiac ion channels by cyclic nucleotide-dependent phosphorylation. In: Sperelakis N. (Ed.) Cell Physiology Sourcebook. Acad. Press, 2012, pp. 431–443. doi: 10.1016/B978-0-12-387738-3.00023-8.
  17. Beavo J.A., Brunton L.L. Cyclic nucleotide research – still expanding after half a century. Nat. Rev. Mol. Cell Biol., 2002, vol. 3, no. 9, pp. 710–717. doi: 10.1038/nrm911.
  18. Gillian B., Brian O.S., Stephen J.Y. Borland G., Smith B.O., Yarwood S.J. EPAC proteins transduce diverse cellular actions of cAMP. Br. J. Pharmacol., 2009, vol. 158, no. 1, pp. 70–86. doi: 10.1111/j.1476-5381.2008.00087.x.
  19. Wróblewski F. The clinical significance of alterations in transaminase activities of serum and other body fluids. Adv. Clin. Chem., 1958, vol. 1, pp. 313–351. doi: 10.1016/S0065-2423(08)60362-5.
  20. Rej R. Aminotransferases in disease. Clin. Lab. Med., 1989, vol. 9, no. 4, pp. 667–687. doi: 10.1016/S0272-2712(18)30598-5.
  21. Dufour D.R., Lott J.A., Nolte F.S., Gretch D.R., Koff R.S. Diagnosis and monitoring of hepatic injury. I. Performance characteristics of laboratory tests. Clin. Chem., 2000, vol. 46, no. 12, pp. 2027–2049. doi: 10.1093/clinchem/46.12.2027.
  22. Kaplan M., Hammerman C. Understanding severe hyperbilirubinemia and preventing kernicterus: Adjuncts in the interpretation of neonatal serum bilirubin. Clin. Chim. Acta, 2005, vol. 356, nos. 1–2, pp. 9–21. doi: 10.1016/j.cccn.2005.01.008.
  23. Vítek L., Ostrow J.D. Bilirubin chemistry and metabolism; harmful and protective aspects. Curr. Pharm. Des., 2009, vol. 15, no. 25, pp. 2869–2883. doi: 10.2174/138161209789058237.
  24. Woreta T.A., Alqahtani S.A. Evaluation of abnormal liver tests. Med. Clin. North Am., 2014, vol. 98, no. 1, pp. 1–16. doi: 10.1016/j.mcna.2013.09.005.
  25. Spinella R., Sawhney R., Jalan R. Albumin in chronic liver disease: Structure, functions and therapeutic implications. Hepatol. Int., 2016, vol. 10, no. 1, pp. 124–132. doi: 10.1007/s12072-015-9665-6.
  26. Matsuura K., Canfield K., Feng W., Kurokawa M. Metabolic regulation of apoptosis in cancer. Int. Rev. Cell Mol. Biol., 2016, vol. 327, pp. 43–87. doi: 10.1016/bs.ircmb.2016.06.006.
  27. Manning B.D., Cantley L.C. AKT/PKB signaling: Navigating downstream. Cell, 2007, vol. 129, no. 7, pp. 1261–1274. doi: 10.1016/j.cell.2007.06.009.
  28. Plas D.R., Thompson C.B. Akt-dependent transformation: There is more to growth than just surviving. Oncogene, 2005, vol. 24, no. 50, pp. 7435–7442. doi: 10.1038/sj.onc.1209097.
  29. Tsujimoto Y. Role of Bcl‐2 family proteins in apoptosis: Apoptosomes or mitochondria? Genes Cells, 1998, vol. 3, no. 11, pp. 697–707. doi: 10.1046/j.1365-2443.1998.00223.x.
  30. Kuida K. Caspase-9. Int. J. Biochem. Cell Biol., 2000, vol. 32, no. 2, pp. 121–124. doi: 10.1016/s1357-2725(99)00024-2.
  31. Liu X., Zou H., Widlak P., Garrard W., Wang X. Activation of the apoptotic endonuclease DFF40 (caspase-activated DNase or nuclease): Oligomerization and direct interaction with histone H1. J. Biol. Chem., 1999, vol. 274, no. 20, pp. 13836–13840. doi: 10.1074/jbc.274.17.11549.
  32. Insel P.A., Zhang L., Murray F., Yokouchi H., Zambon A.C. Cyclic AMP is both a pro-apoptotic and anti-apoptotic second messenger. Acta Physiol., 2012, vol. 204, no. 2, pp. 277–287. doi: 10.1111/j.1748-1716.2011.02273.x.
  33. Harrison F.E., May J.M. Vitamin C function in the brain: Vital role of the ascorbate transporter SVCT2. Free Radicals Biol. Med., 2009, vol. 46, no. 6, pp. 719–730. doi: 10.1016/j.freeradbiomed.2008.12.018.
  34. Manfredini S., Pavan B., Vertuani S., Scaglianti M., Compagnone D., Biondi C., Scatturin A., Tanganelli S., Ferraro L., Prasad P., Dalpiaz A. Design, synthesis and activity of ascorbic acid prodrugs of nipecotic, kynurenic and diclophenamic acids, liable to increase neurotropic activity. J. Med. Chem., 2002, vol. 45, no. 3, pp. 559–562. doi: 10.1021/jm015556r.
  35. Manfredini S., Vertuani S., Pavan B., Vitali F., Scaglianti M., Bortolotti F., Biondi C., Scatturin A., Prasad P., Dalpiaz A. Design, synthesis and in vitro evaluation on HRPE cells of ascorbic and 6-bromoascorbic acid conjugates with neuroactive molecules. Bioorg. Med. Chem., 2004, vol. 12, no. 20, pp. 5453–5463. doi: 10.1016/j.bmc.2004.07.043.
  36. Moteki H., Kimura M., Sunaga, K., Tsuda T., Ogihara M. Signal transduction mechanism for potentiation by α1-and β2-adrenoceptor agonists of L-ascorbic acid-induced DNA synthesis and proliferation in primary cultures of adult rat hepatocytes. Eur. J. Pharmacol., 2013, vol. 700, nos. 1–3, pp. 2–12. doi: 10.1016/j.ejphar.2012.12.010.
  37. Kimura,M., Moteki H., Uchida M., Natsume H., Ogihara M. L-ascorbic acid-and L-ascorbic acid 2-glucoside accelerate in vivo liver regeneration and lower serum alanine aminotransaminase activity in 70% partially hepatectomized rats. Biol. Pharm. Bull., 2014, vol. 37, no. 4, pp. 597–603. doi: 10.1248/bpb.b13-00839.

The content is available under the license Creative Commons Attribution 4.0 License.

Вход в личный кабинет
Ваш логин
Ваш пароль
Забыли пароль? Регистрация