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5 . 2020

Effect of carnosine and α-lipoic acid on hepatocyte apoptosis and the cytokine profile in induced fatty liver disease in Wistar rats

Abstract

Non-alcoholic fatty liver disease (NAFLD) is now a common liver disease affecting about a third of the world’s population. In this regard, the issue of studying the pathogenetic factors of the development of this disease in order to select adequate drug therapy and biologically active substances with antioxidant properties regulating the balance of pro- and anti-inflammatory cytokines is of particular relevance.

The aim of the study was to assess the effect of minor biologically active substances - carnosine and α-lipoic acid on hepatocyte apoptosis and the cytokine profile in the experimental model of the initial stage of NAFLD.

Material and methods. The studies were performed on male Wistar rats with initial body weight of 150±10 g. Animals were divided into 5 groups of 8 rats each. Within 8 weeks, rats of the 1st group (control) received a complete modified diet AIN93M, in which soybean oil was replaced with sunflower oil and lard (1:1). Rats of the experimental groups consumed high-calorie choline-deficient diet (HCCDD), in which fat content was 45%, fructose content - 20% of the energy value of the diet. Rats of the 2nd group were fed HCCDD without any supplements, the 3rd group - with the addition of carnosine (75 mg/kg body weight), the 4th group - with the addition of α-lipoic acid (75 mg/kg body weight), the 5th group - with the addition of carnosine and α-lipoic acid in a total dose of 150 mg/kg body weight. The study of rat hepatocyte apoptosis was performed by flow cytometry. Hepatocytes were stained with annexin V and vital dye 7-aminoactinomycin, followed by detection on an flow cytometer. The content of cytokines and chemokines (IL-1α, IL-10, IL-17А, M-CSF, MIP-1α, MIP-3α, RANTES) in the cytoplasmic fraction of liver tissue was determined by multiplex immunoassay.

Results and discussion. On the model of the initial stage of development of NAFLD in male Wistar rats the еnrichment of HCCDD with carnosine and α-lipoic acid had demonstrated a protective effect on hepatocytes with a decrease in apoptosis intensity to the level in control rats. Under the influence of HCCDD, an increase in the content of M-CSF and MIP-1α and a decrease in the levels of MIP-3α and RANTES, stimulating the migration and differentiation of various immunoregulatory populations to the parenchyma at an early stage of the formation of fatty hepatosis, in the cytoplasmic fraction of liver tissue were detected. Moreover, a decrease in the level of proinflammatory cytokines IL-17A and IL-1α and an increase in IL-10 produced mainly by Treg-populations indicate the absence of pronounced inflammatory changes in the liver of male Wistar rats at the initial stages of development of fatty dystrophy.

Conclusion. Enrichment of HCCDD with both carnosine and а-lipoic acid in Wistar rats had a protective effect on hepatocytes with a decrease in apoptosis to a level in control rats. The increase in the IL-10/IL-17A ratio indicates the activation of anti-inflammatory mechanisms due to the functional predominance of Treg-cells over Th1/Th17 lymphocytes.

Keywords:nonalcoholic fatty liver disease, fatty hepatosis, carnosine, α-lipoic acid, cytokines, chemokines

Funding. The research was carried out at the expense of subsidies for the fulfillment of a state task within the framework of the Program of fundamental scientific research (research topic No. 0529-2019-0058).

Conflict of interests. The authors declare no conflict of interests.

For citation: Trushina E.N., Riger N.A., Mustafina O.K., Timonin AN., Aksenov I.V., Guseva G.V., Tutelyan V.A. Effect of carnosine and α-lipoic acid on hepatocyte apoptosis and the cytokine profile in induced fatty liver disease in Wistar rats. Voprosy pitaniia [Problems of Nutrition]. 2020; 89 (5): 6-16. DOI: https://www.doi.org/10.24411/0042-8833-2020-10061 (in Russian)

Литература

1. Ивашкин В.Т., Драпкина О.М., Маев И.В. и др. Распространенность неалкогольной жировой болезни печени у пациентов амбулаторно-поликлинической практики в Российской Федерации: результаты исследования DIREG 2 // Российский журнал гастроэнтерологии, гепатологии, колопроктологии. 2015. № 6. С. 31–41.

2. Fierbinteanu-Braticevici С., Moldoveanu А., Petrisor А., Diaconu S. Nonalcoholic fatty liver disease: one entity, multiple impacts on liver health // Cell Biol. Toxicol. 2017. Vol. 33. P. 5–14. DOI: https://doi.org/10.1007/s10565-016-9361-x

3. Duvnjak M., Lerotic I., Barcic N. et al. Pathogenesis and management issues for nonalcoholic fatty liver disease // World Gastroenterol. 2007. Vol. 13. P. 4539–4550.

4. Кособян Е.П., Смирнова О.М. Современные концепции патогенеза неалкогольной жировой болезни печени // Сахарный диабет. 2010. № 1. С. 55–64.

5. Ribeiro P.S., Cortez-Pinto H., Solá S. et al. Hepatocyte apoptosis, expression of death receptors, and activation of NF-kappaB in the liver of nonalcoholic and alcoholic steatohepatitis patients // Am. J. Gastroenterol. 2004. Vol. 99, N 9. P. 1708–1717. DOI: https://doi.org/10.1111/j.1572-0241.2004.40009.x

6. Guicciardi M.E., Gores G.J. Apoptosis as a mechanism for liver disease progression // Semin. Liver Dis. 2010. Vol. 30, N 4. P. 402–410. DOI: https://doi.org/10.1055/s-0030-1267540

7. Choi Y., Abdelmegeed M.A. et al. Diet high in fructose promotes liver steatosis and hepatocyte apoptosis in C57BL/6J female mice: Role of disturbed lipid homeostasis and increased oxidative stress // Food Chem. Toxicol. 2017. Vol. 103. P. 111–121. DOI: https://doi.org/10.1016/j.fct.2017.02.039

8. Курбатова И.В., Топчиева Л.В., Дуданова О.П., Шиповская А.А. Биохимические и молекулярно-генетические показатели воспаления и апоптоза при циррозе печени как исходе прогрессирования неалкогольного стеатогепатита // Терапевтический архив. 2019. Т. 91, № 4. С. 21–27. DOI: https://doi.org/10.26442/00403660.2019.04.000057

9. Neuman M.G., Cohen L.B., Nanau R.M. Biomarkers in nonalcoholic fatty liver disease // Can. J. Gastroenterol. Hepatol. 2014. Vol. 28, N 11. P. 607–618. DOI: https://doi.org/10.1155/2014/757929

10. Дуданова О.П., Шиповская А.А., Курбатова И.В. Маркеры печеночно-клеточного повреждения и воспаления при ранней форме неалкогольной жировой болезни печени // Гастроэнтерология Санкт-Петербурга. 2017. № 3. С. 16–20.

11. Braunersreuther V., Viviani G.L., Mach F., Montecucco F. Role of cytokines and chemokines in non-alcoholic fatty liver disease // World J. Gastroenterol. 2012. Vol. 18. P. 727–735.

12. Топчиева Л.В, Курбатова И.В., Дуданова О.П., Соколовская А.А, Шиповская А.А. Полиморфизм генов провоспалительных цитокинов (TNF, IL6) и их рецепторов (TNFRSF1A, TNFRSF1B, IL6R) и неалкогольная жировая болезнь печени // Труды Карельского научного центра РАН. 2017. № 5. С. 3–22. DOI: https://doi.org/10.17076/eb568

13. Neurath M.F., Finotto S. IL-6 signaling in autoimmunity, chronic inflammation-associated cancer // Cytokine Growth Factor Rev. 2011. Vol. 22. P. 83–89. DOI: https://doi.org/10.1016/j.cytogfr.2011.02.003

14. Tilg H., Diehl A.M. Cytokines in alcoholic and nonalcoholic steatohepatitis // N. Engl. J. Med. 2000. Vol. 343. P. 1467–1476.

15. Lake B.G. Preparation and characterization of microsomal fractions for studies on xenobiotic metabolism // Biochemical Toxicology – a Practical Approach / eds K. Snell, B. Mullock. Oxford : IRL Press, 1987. Ch. 8. P. 183–215.

16. Болдырев А.А. Карнозин и защита тканей от окислительного стресса. Москва : Диалог-МГУ, 1999. 362 с.

17. Болдырев А.А., Стволинский С.Л., Федорова Т.Н. Карнозин: эндогенный физиологический корректор активности антиоксидантной системы организма // Успехи физиологических наук. 2007. Т. 38, № 3. С. 57–71.

18. Вишнякова Х.С., Бабижаев М.А., Алипер А.М. и др. Стимуляция пролиферации карнозином: клеточный и транскриптомный подход // Молекулярная биология. 2014. Т. 48, № 5. С. 824–833.

19. Rochette L., Ghibu S., Muresan A., Vergely C. Alpha lipoic acid: molecular mechanisms and therapeutic potential in diabetes // Can. J. Physiol. Pharmacol. 2015. Vol. 93. P. 1021–1027. DOI: https://doi.org/10.1139/cjpp-2014-0353

20. Строков И.А., Фокина А.С. Альфа-липоевая кислота — основное фармакологическое лечение диабетической полинейропатии в стационаре и поликлинике // Журнал неврологии и психиатрии имени С.С. Корсакова. 2017. № 3. С. 50–55.

21. Крыльский Е.Д., Попова Т.Н., Кирилова Е.М., Сафонова О.А. Воздействие липоевой кислоты на активность каспаз, показатели иммунного и антиоксидантного статуса при ревматоидном артрите у крыс // Биоорганическая химия. 2016. Т. 42, № 4. С. 431–439.

22. Lee G.R. The balance of Th17 versus treg cells in autoimmunity // Int. J. Mol. Sci. 2018. Vol. 19, N 3. Article ID E730. DOI: https://doi.org/10.3390/ijms19030730

23. Yi Z., Bishop G.A. Regulatory role of CD40 in obesity-induced insulin resistance // Adipocyte. 2014. Vol. 4, N 1. P. 65–69. DOI: https://doi.org/10.4161/adip.32214

24. Lee B.C., Lee J. Cellular and molecular players in adipose tissue inflammation in the development of obesity-induced insulin resistance // Biochim. Biophys. Acta. 2014. Vol. 1842, N 3. P. 446–462. DOI: https://doi.org/10.1016/j.bbadis.2013.05.017

25. Gotoh K., Inoue M., Masaki T. et al. A novel anti-inflammatory role for spleen-derived interleukin-10 in obesity-induced inflammation in white adipose tissue and liver // Diabetes. 2012. Vol. 61, N 8. P. 1994–2003. DOI: https://doi.org/10.2337/db11-1688

26. Trefts E., Gannon M., Wasserman D.H. The liver // Curr. Biol. 2017. Vol. 27, N 21. P. R1147–R1151. DOI: https://doi.org/10.1016/j.cub.2017.09.019

27. Ma M., Duan R., Zhong H. et al. The crosstalk between fat homeostasis and liver regional immunity in NAFLD // J. Immunol. Res. 2019. Vol. 2019. Article ID 3954890. DOI: https://doi.org/10.1155/2019/3954890

28. Dembic Z. The Cytokines of the Immune System. Cambridge : Academic Press, 2015. 320 p.

29. Hoffmann C., Djerir N., Danckaert A. et al. Hepatic stellate cell hypertrophy is associated with metabolic liver fibrosis // Sci. Rep. 2020. Vol. 10, N 1. P. 3850. DOI: https://doi.org/10.1038/s41598-020-60615-0

30. De Minicis S., Seki E., Uchinami H. et al. Gene expression profiles during hepatic stellate cell activation in culture and in vivo // Gastroenterology. 2007. Vol. 132, N 5. P. 1937–1946.

31. Kashani A., Salehi B., Anghesom D. et al. Spleen size in cirrhosis of different etiologies // J. Ultrasound Med. 2015. Vol. 34, N 2. P. 233–238. DOI: https://doi.org/10.7863/ultra.34.2.233

32. Li L., Duan M., Chen W. et al. The spleen in liver cirrhosis: revisiting an old enemy with novel targets // J. Transl. Med. 2017. Vol. 15, N 1. P. 111–120.

33. Wang K.C., Tsai C.P., Lee C.L. et al. α-Lipoic acid enhances endogenous peroxisome-proliferator-activated receptor-γ to ameliorate experimental autoimmune encephalomyelitis in mice // Clin. Sci. (Lond.). 2013. Vol. 125, N 7. P. 329–340. DOI:https://doi.org/10.1042/CS20120560

34. Stec D.E., Gordon D.M., Hipp J.A. et al. Loss of hepatic PPARα promotes inflammation and serum hyperlipidemia in diet-induced obesity // Am. J. Physiol. Regul. Integr. Comp. Physiol. 2019. Vol. 317, N 5. P. R733–R745. DOI:https://doi.org/10.1152/ajpregu.00153.2019

35. Forsberg E.A., Botusan I.R., Wang J. et al. Carnosine decreases IGFBP1 production in db/db mice through suppression of HIF-1 // J. Endocrinol. 2015. Vol. 225, N 3. P. 159–167. DOI: https://doi.org/10.1530/JOE-14-0571

36. Shi L., Banerjee D., Dobierzewska A. et al. Direct regulation of IGF-binding protein 1 promoter by interleukin-1β via an insulin- and FoxO-1-independent mechanism // Am. J. Physiol. Endocrinol. Metab. 2016. Vol. 310, N 8. P. E612–E623. DOI:https://doi.org/10.1152/ajpendo.00289.2015

References

1. Ivashkin V.T., Drapkina O.M., Maev I.V., et al. The prevalence of non-alcoholic fatty liver disease in patients with outpatient practice in the Russian Federation: the results of the DIREG 2 study. Rossiyskiy zhurnal gastroenterologii, gepatologii, koloproktologii [Russian Journal of Gastroenterology, Hepatology, Coloproctology]. 2015; (6): 31–41. (in Russian)

2. Fierbinteanu-Braticevici С., Moldoveanu А., Petrisor А., Diaconu S. Nonalcoholic fatty liver disease: one entity, multiple impacts on liver health. Cell Biol Toxicol. 2017; 33: 5–14. DOI: https://doi.org/10.1007/s10565-016-9361-x

3. Duvnjak M., Lerotic I., Barcic N., et al. Pathogenesis and management issues for nonalcoholic fatty liver disease. World Gastroenterol. 2007; 13: 4539–50.

4. Kosobyan E.P., Smirnova O.M. Modern concepts of the pathogenesis of non-alcoholic fatty liver disease. Sakharniy diabet [Diabetes Mellitus]. 2010; (1): 55–64. (in Russian)

5. Ribeiro P.S., Cortez-Pinto H., Solá S., et al. Hepatocyte apoptosis, expression of death receptors, and activation of NF-kappaB in the liver of nonalcoholic and alcoholic steatohepatitis patients. Am J Gastroenterol. 2004; 99 (9): 1708–17. DOI: https://doi.org/10.1111/j.1572-0241.2004.40009.x

6. Guicciardi M.E., Gores G.J. Apoptosis as a mechanism for liver disease progression. Semin Liver Dis. 2010; 30 (4): 402–10. DOI: https://doi.org/10.1055/s-0030-1267540

7. Choi Y., Abdelmegeed M.A., et al. Diet high in fructose promotes liver steatosis and hepatocyte apoptosis in C57BL/6J female mice: Role of disturbed lipid homeostasis and increased oxidative stress. Food Chem Toxicol. 2017; 103: 111–21. DOI: https://doi.org/10.1016/j.fct.2017.02.039

8. Kurbatova I.V., Topchieva L.V., Dudanova O.P., Shipovskaya A.A. Biochemical and molecular genetic indicators of inflammation and apoptosis in liver cirrhosis as an outcome of the progression of non-alcoholic steatohepatitis. Terapevticheskiy arkhiv [Therapeutic Archive]. 2019; 91 (4): 21–7. DOI: https://doi.org/10.26442/00403660.2019.04.000057 (in Russian)

9. Neuman M.G., Cohen L.B., Nanau R.M. Biomarkers in nonalcoholic fatty liver disease. Can J Gastroenterol Hepatol. 2014; 28 (11): 607–18. DOI: https://doi.org/10.1155/2014/757929

10. Dudanova O.P., Shipovskaya A.A., Kurbatova I.V. Markers of hepatic cell damage and inflammation in the early form of non-alcoholic fatty liver disease. Gastroenterologiya Sankt-Peterburga [Gastroenterology of Saint Petersburg]. 2017; (3): 16–20. (in Russian)

11. Braunersreuther V., Viviani G.L., Mach F., Montecucco F. Role of cytokines and chemokines in non-alcoholic fatty liver disease. World J Gastroenterol. 2012; 18: 727–35.

12. Topchieva L.V., Kurbatova I.V., Dudanova O.P., Sokolovskaya A.A., Shipovskaya A.A. Gene polymorphism of pro-inflammatory cytokines (TNF, IL6) and their receptors (TNFRSF1A, TNFRSF1B, IL6R) and non-alcoholic fatty liver disease. Trudy Karel’skogo nauchnogo tsentra RAN [Transactions of the Karelian Scientific Center of the Russian Academy of Sciences]. 2017; (5): 3–22. DOI: https://doi.org/10.17076/eb568 (in Russian)

13. Neurath M.F., Finotto S. IL-6 signaling in autoimmunity, chronic inflammation-associated cancer. Cytokine Growth Factor Rev. 2011; 22: 83–9. DOI: https://doi.org/10.1016/j.cytogfr.2011.02.003

14. Tilg H., Diehl A.M. Cytokines in alcoholic and nonalcoholic steatohepatitis. N Engl J Med. 2000; 343: 1467–76.

15. Lake B.G. Preparation and characterization of microsomal fractions for studies on xenobiotic metabolism. In: K. Snell, B. Mullock (eds). Biochemical Toxicology – a Practical Approach. Oxford: IRL Press, 1987; 8: 183–215.

16. Boldyrev A.A. Carnosine and the protection of tissues from oxidative stress. Moscow: Dialog-MGU, 1999: 362 p. (in Russian)

17. Boldyrev A.A., Stvolinskiy S.L., Fedorova T.N. Carnosine: an endogenous physiological corrector of the activity of the antioxidant system of the body. Uspekhi fiziologicheskikh nauk [Advances of the Physiological Sciences]. 2007; 38 (3): 57–71. (in Russian)

18. Vishnyakova Kh.S., Babizhaev M.A., Aliper A.M., et al. Stimulation of proliferation by carnosine: a cellular and transcriptomic approach. Molekulyarnaya biologiya [Molecular Biology]. 2014; 48 (5): 824–33. (in Russian)

19. Rochette L., Ghibu S., Muresan A., Vergely C. Alpha lipoic acid: molecular mechanisms and therapeutic potential in diabetes. Can J Physiol Pharmacol. 2015; 93: 1021–7. DOI: https://doi.org/10.1139/cjpp-2014-0353

20. Strokov I.A., Fokina A.S. Alpha-lipoic acid is the main pharmacological treatment of diabetic polyneuropathy in a hospital and clinic. Zhurnal nevrologii i psikhiatrii imeni S.S. Korsakova [Korsakov’s Journal of Neurology and Psychiatry]. 2017; (3): 50–5. (in Russian)

21. Kryl’skiy E.D., Popova T.N., Kirilova E.M., Safonova O.A. The effect of lipoic acid on caspase activity, indicators of the immune and antioxidant status in rat rheumatoid arthritis. Bioorganicheskaya khimiya [Bioorganic Chemistry. 2016; 42 (4): 431–9. (in Russian)

22. Lee G.R. The balance of Th17 versus treg cells in autoimmunity. Int J Mol Sci. 2018; 19 (3): E730. DOI:https://doi.org/10.3390/ijms19030730

23. Yi Z., Bishop G.A. Regulatory role of CD40 in obesity-induced insulin resistance. Adipocyte. 2014; 4 (1) : 65–9. DOI:https://doi.org/10.4161/adip.32214

24. Lee B.C., Lee J. Cellular and molecular players in adipose tissue inflammation in the development of obesity-induced insulin resistance. Biochim Biophys Acta. 2014; 1842 (3): 446–62. DOI:https://doi.org/10.1016/j.bbadis.2013.05.017

25. Gotoh K., Inoue M., Masaki T., et al. A novel anti-inflammatory role for spleen-derived interleukin-10 in obesity-induced inflammation in white adipose tissue and liver. Diabetes. 2012; 61 (8): 1994–2003. DOI:https://doi.org/10.2337/db11-1688

26. Trefts E., Gannon M., Wasserman D.H. The liver. Curr Biol. 2017; 27 (21): R1147–51. DOI: https://doi.org/10.1016/j.cub.2017.09.019

27. Ma M., Duan R., Zhong H., et al. The crosstalk between fat homeostasis and liver regional immunity in NAFLD. J Immunol Res. 2019; 2019: 3954890. DOI: https://doi.org/10.1155/2019/3954890

28. Dembic Z. The Cytokines of the Immune System. Cambridge: Academic Press, 2015. 320 p.

29. Hoffmann C., Djerir N., Danckaert A., et al. Hepatic stellate cell hypertrophy is associated with metabolic liver fibrosis. Sci Rep. 2020; 10 (1): 3850. DOI:https://doi.org/10.1038/s41598-020-60615-0

30. De Minicis S., Seki E., Uchinami H., et al. Gene expression profiles during hepatic stellate cell activation in culture and in vivo. Gastroenterology. 2007; 132 (5): 1937–46.

31. Kashani A., Salehi B., Anghesom D., et al. Spleen size in cirrhosis of different etiologies. J Ultrasound Med. 2015; 34 (2): 233–8. DOI: https://doi.org/10.7863/ultra.34.2.233

32. Li L., Duan M., Chen W., et al. The spleen in liver cirrhosis: revisiting an old enemy with novel targets. J Transl Med. 2017; 15 (1): 111–20.

33. Wang K.C., Tsai C.P., Lee C.L., et al. α-Lipoic acid enhances endogenous peroxisome-proliferator-activated receptor-γ to ameliorate experimental autoimmune encephalomyelitis in mice. Clin Sci (Lond). 2013; 125 (7): 329–40. DOI: https://doi.org/10.1042/CS20120560

34. Stec D.E., Gordon D.M., Hipp J.A., et al. Loss of hepatic PPARα promotes inflammation and serum hyperlipidemia in diet-induced obesity. Am J Physiol Regul Integr Comp Physiol. 2019; 317 (5): R733–45. DOI:https://doi.org/10.1152/ajpregu.00153.2019

35. Forsberg E.A., Botusan I.R., Wang J., et al. Carnosine decreases IGFBP1 production in db/db mice through suppression of HIF-1. J Endocrinol. 2015; 225 (3): 159–67. DOI: https://doi.org/10.1530/JOE-14-0571

36. Shi L., Banerjee D., Dobierzewska A., et al. Direct regulation of IGF-binding protein 1 promoter by interleukin-1β via an insulin- and FoxO-1-independent mechanism. Am J Physiol Endocrinol Metab. 2016; 310 (8): E612–23. DOI:https://doi.org/10.1152/ajpendo.00289.2015

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CHIEF EDITOR
CHIEF EDITOR
Viktor A. Tutelyan
Full Member of the Russian Academy of Sciences, Doctor of Medical Sciences, Professor, Scientific Director of the Federal Research Centre of Nutrition, Biotechnology and Food Safety (Moscow, Russia)
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