Palmitat ile Non-alkolik Yağlı Karaciğer Hastalığı Modeli Oluşturulan HepG2 Hücrelerinde Rosmarinik Asitin Hücre Canlılığına, Yağlanmaya, Paraoksonaz-1 ve Paraoksonaz-3 Protein Düzeylerine Etkisi

Amaç: Palmitat ile non-alkolik yağlı karaciğer hastalığı modeli oluşturulan HepG2 hücrelerinde rosmarinik asitin (RA) hücre canlılığına, yağlanmaya, paraoksonaz (PON) 1 ve PON3 protein düzeylerine etkisini araştırmayı amaçladık. Gereç ve Yöntem: Deneysel yağlanma modeli oluşturmak için, HepG2 hücreleri 1 mM palmitat ile 24 saat inkübe edildi. Tedavi olarak palmitat ile aynı anda hücre kültürü medyumuna, HepG2 hücrelerine toksik olmayan RA konsantrasyonları eklendi. Hücre canlılığı 3-(4,5-dimetil-2-tiazolil)-2,5- difenil-2H-tetrazolium bromür testi ile değerlendirildi. Yağlanmanın değerlendirilmesi için hücre içi trigliserid düzeyleri ölçüldü ve hücreler Oil-Red O ile boyanarak mikroskopik olarak incelendi. PON1 ve PON3 protein düzeyleri Western blot yöntemiyle ölçüldü. Bulgular: 1 mM palmitat hücre canlılığında anlamlı bir azalmaya ve trigliserid düzeylerinde anlamlı bir artışa yol açtı, PON1 ve PON3 protein düzeylerini ise anlamlı olarak değiştirmedi. Palmitat ile oluşturulan deneysel non-alkolik yağlı karaciğer hastalığı (NAFLD) modelinde RA, hücre canlılığını anlamlı olarak artırdı ve trigliserid düzeylerini anlamlı olarak azalttı. Oil-Red O ile boyanan hücrelerin mikroskopik incelenmelerinde hücrelerin trigliserid düzeylerindeki değişimler ile benzer bulgular görüldü. RA hem palmitat uygulanmayan hem de palmitat uygulanan HepG2 hücrelerinde PON1 ve PON3 protein düzeylerini anlamlı olarak değiştirmedi. Sonuç: Çalışmamız, RA’nın palmitat ile NAFLD modeli oluşturulan HepG2 hücrelerinde hücre canlılığını artırdığını, yağlanmayı azalttığını, ancak PON1 ve PON3 protein düzeylerini değiştirmediğini gösterdi.

The Effect of Rosmarinic Acid on Cell Viability, Steatosis, Paraoxonase-1, and Paraoxonase-3 Protein Levels in Palmitateinduced Non-alcoholic Fatty Liver Disease Model in HepG2 Cells

Aim: We aimed to investigate the effect of rosmarinic acid (RA) on cell viability, steatosis, paraoxonase (PON)1, and PON3 protein levels inpalmitate-induced non-alcoholic fatty liver disease (NAFLD) model in HepG2 cells.Materials and Methods: To induce an experimental steatosis model, HepG2 cells were incubated with 1 mM palmitate for 24 hours. For thetreatment, non-toxic RA concentrations were added to the cell culture medium simultaneously with the palmitate. Cell viability was evaluated by3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. To evaluate steatosis, intracellular triglyceride levels were measured andthe cells were examined microscopically with Oil-Red O staining. PON1 and PON3 protein levels were measured by Western blotting.Results: 1 mM palmitate caused a significant decrease in cell viability and a significant increase in triglyceride levels, but it did not significantlychange PON1 and PON3 protein levels. RA caused a significant increase in cell viability and a significant decrease in triglyceride levels in thepalmitate-treated cells. Similar findings with the triglyceride levels of cells were shown in microscopic examination of cells that were stained withOil-Red O. RA did not significantly change PON1 and PON3 protein levels in neither non-treated cells nor treated cells with palmitate.Conclusion: Our study showed that RA increases cell viability and decreases steatosis, but it does not change PON1 and PON3 protein levels inpalmitate-induced NAFLD model in HepG2 cells.

PDF

___

1. Chalasani N, Younossi Z, Lavine JE, Diehl AM, Brunt EM, Cusi K, et al. The diagnosis and management of non-alcoholic fatty liver disease: practice Guideline by the American Association for the Study of Liver Diseases, American College of Gastroenterology, and the American Gastroenterological Association. Hepatology. 2012;55:2005-23.
2. DeFilippis AP, Blaha MJ, Martin SS, Reed RM, Jones SR, Nasir K, et al. Nonalcoholic fatty liver disease and serum lipoproteins: the Multi-Ethnic Study of Atherosclerosis. Atherosclerosis. 2013;227:429-36.
3. Araya J, Rodrigo R, Videla LA, Thielemann L, Orellana M, Pettinelli P et al. Increase in long-chain polyunsaturated fatty acid n - 6/n - 3 ratio in relation to hepatic steatosis in patients with non-alcoholic fatty liver disease. Clin Sci (Lond). 2004;106:635-43.
4. Byrne CD, Targher G. NAFLD: a multisystem disease. J Hepatol. 2015;62(1 Suppl):47-64.
5. Sookoian S, Pirola CJ. Non-alcoholic fatty liver disease is strongly associated with carotid atherosclerosis: a systematic review. J Hepatol. 2008;49:600-7.
6. Précourt LP, Amre D, Denis MC, Lavoie JC, Delvin E, Seidman E, et al. The three-gene paraoxonase family: physiologic roles, actions and regulation. Atherosclerosis. 2011;214:20-36.
7. Gómez-Lechón MJ, Donato MT, Martínez-Romero A, Jiménez N, Castell JV, O’Connor JE. A human hepatocellular in vitro model to investigate steatosis. Chem Biol Interact. 2007;165:106-16.
8. Özgün GS, Özgün E, Tabakçıoğlu K, Gökmen SS, Eskiocak S. Effect of palmitate-induced steatosis on paraoxonase-1 and paraoxonase-3 enzymes in human-derived liver (HepG2) cells. Archives of Clinical and Experimental Medicine. 2019;4:118-21.
9. Jang E, Shin MH, Kim KS, Kim Y, Na YC, Woo HJ, et al. Anti-lipoapoptotic effect of Artemisia capillaris extract on free fatty acids-induced HepG2 cells. BMC Complement Altern Med. 2014;14:253.
10. Kim M, Yoo G, Randy A, Son YJ, Hong CR, Kim SM, et al. Lemon Balm and Its Constituent, Rosmarinic Acid, Alleviate Liver Damage in an Animal Model of Nonalcoholic Steatohepatitis. Nutrients. 2020;12:1166.
11. Bulgakov VP, Inyushkina YV, Fedoreyev SA. Rosmarinic acid and its derivatives: biotechnology and applications. Crit Rev Biotechnol. 2012;32:203-17.
12. Kim GD, Park YS, Jin YH, Park CS. Production and applications of rosmarinic acid and structurally related compounds. Appl Microbiol Biotechnol. 2015;99:2083-92.
13. Park JY, Kim Y, Im JA, Lee H. Oligonol suppresses lipid accumulation and improves insulin resistance in a palmitate-induced in HepG2 hepatocytes as a cellular steatosis model. BMC Complement Altern Med. 2015;15:185.
14. Xu S, Nam SM, Kim JH, Das R, Choi SK, Nguyen TT, et al. Palmitate induces ER calcium depletion and apoptosis in mouse podocytes subsequent to mitochondrial oxidative stress. Cell Death Dis. 2015;6:e1976.
15. Yang X, Chan C. Repression of PKR mediates palmitate-induced apoptosis in HepG2 cells through regulation of Bcl-2. Cell Res. 2009;19:469-86.
16. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983;65:55-63.
17. Ahmadian S, Barar J, Saei AA, Fakhree MA, Omidi Y. Cellular toxicity of nanogenomedicine in MCF-7 cell line: MTT assay. J Vis Exp. 2009;(26):1191.
18. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193:265-75.
19. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680-5.
20. Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 2012;9:671-5.
21. Younossi ZM, Koenig AB, Abdelatif D, Fazel Y, Henry L, Wymer M. Global epidemiology of nonalcoholic fatty liver disease-Meta-analytic assessment of prevalence, incidence, and outcomes. Hepatology. 2016;64:73-84.
22. Review Team, LaBrecque DR, Abbas Z, Anania F, Ferenci P, Khan AG, et al. World Gastroenterology Organisation global guidelines: Nonalcoholic fatty liver disease and nonalcoholic steatohepatitis. J Clin Gastroenterol. 2014;48:467-73.
23. Pérez-Fons L, Garzón MT, Micol V. Relationship between the antioxidant capacity and effect of rosemary (Rosmarinus officinalis L.) polyphenols on membrane phospholipid order. J Agric Food Chem. 2010;58:161-71.
24. Polimeni L, Del Ben M, Baratta F, Perri L, Albanese F, Pastori D, et al. Oxidative stress: New insights on the association of non-alcoholic fatty liver disease and atherosclerosis. World J Hepatol. 2015;7:1325-36.
25. Adomako-Bonsu AG, Chan SL, Pratten M, Fry JR. Antioxidant activity of rosmarinic acid and its principal metabolites in chemical and cellular systems: Importance of physico-chemical characteristics. Toxicol In Vitro. 2017;40:248-55.
26. Wu J, Zhu Y, Li F, Zhang G, Shi J, Ou R, et al. Spica prunellae and its marker compound rosmarinic acid induced the expression of efflux transporters through activation of Nrf2-mediated signaling pathway in HepG2 cells. J Ethnopharmacol. 2016;193:1-11.
27. Ozgun GS, Ozgun E. The cytotoxic concentration of rosmarinic acid increases MG132-induced cytotoxicity, proteasome inhibition, autophagy, cellular stresses, and apoptosis in HepG2 cells. Hum Exp Toxicol. 2020;39:514-23.
28. Ma ZJ, Yan H, Wang YJ, Yang Y, Li XB, Shi AC, et al. Proteomics analysis demonstrating rosmarinic acid suppresses cell growth by blocking the glycolytic pathway in human HepG2 cells. Biomed Pharmacother. 2018;105:334-49.
29. Lima CF, Fernandes-Ferreira M, Pereira-Wilson C. Phenolic compounds protect HepG2 cells from oxidative damage: relevance of glutathione levels. Life Sci. 2006;79:2056-68.
30. Kudchodkar BJ, Lacko AG, Dory L, Fungwe TV. Dietary fat modulates serum paraoxonase 1 activity in rats. J Nutr. 2000;130:2427-33.
31. Boshtam M, Razavi AE, Pourfarzam M, Ani M, Naderi GA, Basati G, et al. Serum paraoxonase 1 activity is associated with fatty acid composition of high density lipoprotein. Dis Markers. 2013;35:273-80.