OKSİRESVERATROLÜN L929 FİBROBLAST HÜCRELERİNDEKİ OKSİDATİF STRES HASARI ÜZERİNE OLUMLU ETKİLERİ

Abstract

Amaç: L929 fibroblast hücrelerinde H2O2 etkisiyle oluşturulan hücre hasarının bertaraf edilmesinde oksiresveratrolün rejeneratif etkisinin değerlendirilmesi amaçlanmıştır. Gereç ve Yöntem: Oksiresveratrolün total antioksidan seviyesi (TAS), total oksidan seviyesi (TOS), oksidatif stres indeksi (OSİ), toplam fenolik madde seviyeleri farklı derişimlerde ölçülmüştür. Oksiresveratrol fibroblast hücrelerinde IC50 değeri MTT yöntemiyle belirlenmesinin ardından 12.5 - 400 µM konsantrasyonlarında H2O2 ile meydana getirilen oksidatif stres hasarındaki rejeneratif etkisi in-vitro ortamdaki hücre proliferasyonunun ölçümü xCELLigence cihazıyla gerçekleştirilmiştir. Ayrıca hücre hasarındaki yara iyileşme düzeylerini belirlemek amacıyla 3.125 - 25 µM derişimlerinde scratch analizi yapılmıştır. Sonuç ve Tartışma: Oksiresveratrolün 0.5 mg/ml’de TAS değeri 3 ± 0.3 ve TOS değeri 0.77 ± 0.52; (OSİ) değeri 0.02 ± 0.09 olarak bulunmuştur. Oksiresveratrolün 0.625 mg/ml’den 10 mg/ml’ye farklı dozlarındaki total fenolik madde (TFM) derişimleri düşük konsantrasyondan yüksek konsantrasyona daha yüksek TFM’ye sahip olduğu gözlemlenmiştir. Oksiresveratrolün L929 fibroblast hücreleri IC50 değeri 214.2 μM; H2O2’e maruz bırakılan L929 fibroblast hücrelerindeki hücre hattında oksiresveratrol IC50 değeri 109.7 μM olduğu belirlenmiştir. Oksiresveratrolün 12.5 μM konsantrasyonunun xCELLigence cihazıyla ölçülmesinde en fazla hücre proliferasyonu gözlenmiştir. Scratch analizinde H2O2 ile hasarlanmış hücrelerdeki 12.5 μM oksiresveratrol konsantrasyonunda 24. Saat için %62, 48. Saat için %88’lik bir hücre proliferasyonuyla diğer dozlardan daha fazla iyileşme gözlenmiştir. 12.5 μM konsantrasyonu hem proliferasyon, hem de scratch analizinde en etkili konsantrasyon olduğu belirlenmiştir. Oksiresveratrol; düşük konsantrasyonlarda gösterdiği antioksidan kapasiteyle H2O2 aracılığıyla meydana getirilen hücresel hasarda, L929 fibroblast hücrelerini oksidatif stresten korur, fibroblast canlılığına ve migrasyona bir güç çarpanı etkisi oluşturur. Bu sonuçlar oksiresveratrolün; serbest oksijen radikallerinin oluşturduğu akut ya da kronik hastalıkların engellenmesinde hücre düzeyinde etkili olabileceğini göstermiştir. Hücre düzeyinde gerçekleştirilecek daha kapsamlı çalışmalar, in-vivo çalışmalarla oksidatif stres kaynaklı hastalıkların tedavisinde oksiresveratrolün potansiyel bir molekül olabileceği düşünülebilir.

Keywords

• Oksiresveratrol
• OSİ
• TAS
• TFM
• TOS

POSITIVE EFFECTS OF OXYRESVERATROL ON OXIDATIVE STRESS DAMAGE IN L929 FIBROBLAST CELLS

Abstract

Objective: It was aimed to determine the regenerative effect of oxyresveratrol in eliminating the cell damage caused by the effect of H2O2 in L929 fibroblast cells. Material and Method: Total antioxidant capacity (TAC), total oxidant capacity (TOC), oxidative stress index (OSI), total phenolic substance levels of oxyresveratrol were measured at different concentrations. After the IC50 value of oxyresveratrol fibroblast cells was determined by the MTT method, the regenerative effect on oxidative stress damage induced by H2O2 at 12.5 - 400 µM concentrations was performed with the xCELLigence device to measure cell proliferation in-vitro. In addition, scratch analysis was performed at concentrations of 3.125 - 25 µM in order to determine the wound healing levels in cell damage. Result and Discussion: The TAC value of oxyresveratrol at 0.5 mg/ml was 3 ± 0.3 and the TOC value was 0.77 ± 0.52; (OSI) value was found to be 0.02 ± 0.09. It has been observed that the total phenolic substance (TPS) concentrations of oxiresveratrol at different doses from 0.625 mg/ml to 10 mg/ml have higher TPS from low concentration to high concentration. Oxyresveratrol L929 fibroblast cells IC50 value 214.2 μM; The IC50 value of oxyresveratrol was determined to be 109.7 μM in the cell line of L929 fibroblast cells exposed to H2O2. The highest cell proliferation was observed when measuring the 12.5 μM concentration of oxiresveratrol with the xCELLigence device. Scratch analysis showed a greater improvement than other doses, with a cell proliferation of 62% at the 24th hour and 88% at the 48th hour at a concentration of 12.5 μM oxyresveratrol in H2O2-damaged cells. The 12.5 μM concentration was determined to be the most effective concentration in both proliferation and scratch analysis. Oxyresveratrol; with its antioxidant capacity at low concentrations, L929 protects fibroblast cells from oxidative stress in cellular damage caused by H2O2, and creates a power multiplier effect on fibroblast viability and migration. These results indicate that oxyresveratrol; showed that it can be effective at the cellular level preventing of acute or chronic diseases caused by free oxygen radicals. It can be thought that oxyresveratrol may be a potential molecule in the treatment of oxidative stress-induced diseases with more comprehensive studies to be carried out at the cellular level and in-vivo studies.

Keywords

• OSI
• oxyresveratrol
• TAC
• TPS
• TOC

References

1. 1. Yan, L.J., Sohal, R.S. (1998). Mitochondrial adenine nucleotide translocase is modified oxidatively during aging. Proceedings of the National Academy of Sciences, 95(22), 12896-12901. [CrossRef]
2. 2. Yan, L.J. (2014). Positive oxidative stress in aging and aging-related disease tolerance. Redox Biology, 2, 165-169. [CrossRef]
3. 3. Dokuyucu, R., Karateke, A., Gokce, H., Kurt, R.K., Ozcan, O., Ozturk, S., Tas, Z.A., Karateke, F., Duru, M. (2014). Antioxidant effect of erdosteine and lipoic acid in ovarian ischemia-reperfusion injury. European Journal of Obstetrics & Gynecology and Reproductive Biology, 183, 23-27. [CrossRef]
4. 4. Berlett, B.S., Stadtman, E.R. (1997). Protein oxidation in aging, disease, and oxidative stress. Journal of Biological Chemistry, 272(33), 20313-20316. [CrossRef]
5. 5. Motor, S., Öztürk, S., Özcan, O., Gürpinar, A.B., Can, Y., Yuksel, R., Yenin, J.Z., Seraslan, G., Öztürk, O.H. (2014). Evaluation of total antioxidant status, total oxidant status and oxidative stress index in patients with alopecia areata. International Journal of Clinical and Experimental Medicine, 7(4), 1089-1093.
6. 6. Aydın, M., Selcoki, Y., Nazlı, Y., Çolak, N., Serkan Yalçın, K., Canbal, M., Demirçelik, B., Yiğitoğlu, R., Eryonucu, B. (2012). Relationship between total antioxidant capacity and the severity of coronary artery disease. Journal of Clinical & Experimental Investigations, 3(1), 22-28. [CrossRef]
7. 7. Şahin, D.Y., Elbasan, Z., Gür, M., Türkoğlu, C., Özaltun, B., Sümbül, Z., Çaylı, M. (2012). Relationship between oxidative stress markers and cardiac syndrome X. Journal of Clinical & Experimental Investigations, 3(2), 174-180. [CrossRef]
8. 8. Kähkönen, M.P., Hopia, A.I., Vuorela, H.J., Rauha, J.P., Pihlaja, K., Kujala, T.S., Heinonen, M. (1999). Antioxidant activity of plant extracts containing phenolic compounds. Journal of Agricultural and Food Chemistry, 47(10), 3954-3962. [CrossRef]

9. 9. Kay, C.D. (2010). The future of flavonoid research. British Journal of Nutrition, 104(S3), 91-95. [CrossRef]
10. 10. Hooper, L., Kroon, P.A., Rimm, E.B., Cohn, J.S., Harvey, I., Le Cornu, K.A., Ryder, J.J., Hall, W.L., Cassidy, A. (2008). Flavonoids, flavonoid-rich foods, and cardiovascular risk: a meta-analysis of randomized controlled trials. The American Journal of Clinical Nutrition, 88(1), 38-50. [CrossRef]
11. 11. Güvenç, M. (2008). PhD Thesis. The effects of resveratrol, lipoic acid and vitamin C on lipophilic vitamins, cholesterol and fatty acid composition in liver, kidney and erythrocytes of type-1 diabetic rats. Department of Pharmaceutical Biology, Institute of Science, Fırat University, Elazığ, Turkey.
12. 12. Altınışık, M., (2000). Serbest oksijen radikalleri ve antioksidanlar. Erişim adresi https://www.mustafaaltinisik.org.uk/21-adsem-01s.pdf. Erişim tarihi: 05.04.2023.
13. 13. Gupta, A.K., Rather, M.A., Kumar, J.A., Shashank, A., Singhal, S., Sharma, M., Mastinu, A. (2020). Artocarpus lakoocha roxb. and artocarpus heterophyllus lam. flowers: New sources of bioactive compounds. Plants, 9(10), 1329. [CrossRef]
14. 14. Maneechai, S., Likhitwitayawuid, K., Sritularak, B., Palanuvej, C., Ruangrungsi, N., Sirisa-Ard, P. (2009). Quantitative analysis of oxyresveratrol content in Artocarpus lakoocha and ‘Puag-Haad’. Medical Principles and Practice, 18(3), 223-227. [CrossRef]
15. 15. Wikipedia Web site. (2021). from https://tr.wikipedia.org/wiki/Beyaz_dut. Erişim tarihi: 07.04.2021.
16. 16. Kim, J.K., Kim, M., Cho, S.G., Kim, M.K., Kim, S.W., Lim, Y.H. (2010). Biotransformation of mulberroside A from Morus alba results in enhancement of tyrosinase inhibition. Journal of Industrial Microbiology and Biotechnology, 37(6), 631-637. [CrossRef]
17. 17. Kim, Y.M., Yun, J., Lee, C.K., Lee, H., Min, K.R., Kim, Y. (2002). Oxyresveratrol and hydroxystilbene compounds: inhibitory effect on tyrosinase and mechanism of action. Journal of Biological Chemistry, 277(18), 16340-16344. [CrossRef]
18. 18. Hu, S., Chen, F., Wang, M. (2015). Photoprotective effects of oxyresveratrol and Kuwanon O on DNA damage induced by UVA in human epidermal keratinocytes. Chemical Research in Toxicology, 28(3), 541-548. [CrossRef]
19. 19. Radapong, S., Sarker, S.D., Ritchie, K.J. (2020). Oxyresveratrol possesses DNA damaging activity. Molecules, 25(11), 2577. [CrossRef]
20. 20. Likhitwitayawuid K., Sritularak B., Benchanak K., Lipipun V., Mathew J., Schinazi R.F. (2005). Phenolics with antiviral activity from Millettia erythrocalyx and Artocarpus lakoocha. Natural Product Research, 19, 177-182. [CrossRef]
21. 21. Likhitwitayawuid K., Supudompol, B., Sritularak, B., Lipipun, V., Rapp, K., Schinazi, R.F. (2005). Phenolics with anti-HSV and anti-HIV activities from Artocarpus gomezianus, Mallotus pallidus and Triphasia trifoliat. Pharmaceutical Biology, 43, 651-657. [CrossRef]
22. 22. Chuanasa, T., Phromjai, J., Lipipun, V., Likhitwitayawuid, K., Suzuki, M., Pramyothin P., Hattori, M., Shiraki, K. (2008). Anti-herpes simplex virus activity of oxyresveratrol derived from Thai medicinal plant: mechanism of action and therapeutic efficacy on cutaneous infection in mice. Antiviral Research, 80, 62-70. [CrossRef]
23. 23. Lorenz, P., Roychowdhury, S., Engelmann, M., Wolf, G., Horn, T.F.W. (2003). Oxyresveratrol and resveratrol are potent antioxidants and free radical scavengers: effect on nitrosative and oxidative stress derived from microglial cells. Nitric Oxide, 9, 64-76. [CrossRef]
24. 24. Andrabi, S.A., Spina, M.G., Lorenz, P., Ebmeyer, U., Wolf, G., Horn, T.F.W. (2004). Oxyresveratrol (trans -2,3,4,5-tetrahydroxystilbene) is neuroprotective and inhibits the apoptotic cell death in transient cerebral ischemia. Brain Research, 1017, 98-107. [CrossRef]
25. 25. Breuer, C., Wolf, G., Andrabi, S.A., Lorenz, P., Horn, T.F.W. (2006). Blood-brain barrier permeability to the neuroprotectant oxyresveratrol. Neuroscience Letter, 393, 113-118. [CrossRef]
26. 26. Sunilkumar, D., Drishya, G., Chandrasekharan, A., Shaji, S.K., Bose, C., Jossart, J., Nair, B.G. (2020). Oxyresveratrol drives caspase-independent apoptosis-like cell death in MDA-MB-231 breast cancer cells through the induction of ROS. Biochemical Pharmacology, 173, 113724. [CrossRef]
27. 27. Erel, O. (2004). A novel automated direct measurement method for total antioxidant capacity using a new generation, more stable ABTS radical cation. Clinical Biochemistry, 37(4), 277-285. [CrossRef]
28. 28. Erel, O. (2005). A new automated colorimetric method for measuring total oxidant status. Clinical Biochemistry, 38(12), 1103-1111. [CrossRef]
29. 29. Celal, B., Sevindik, M., Akgül, H., Selamoglu, Z. (2019). Oxidative stress index and antioxidant capacity of Lepista nuda collected from Gaziantep/Turkey. Sigma, 37(1), 1-5.
30. 30. Büyüktuncel, E. (2013). Toplam fenolik içerik ve antioksidan kapasite tayininde kullanılan başlıca spektrofotometrik yöntemler. Marmara Pharmaceutical Journal, 17(2), 93-103.
31. 31. Slinkard, K., Singleton, V.L. (1977). Total phenol analysis: automation and comparison with manual methods. American Journal of Enology and Viticulture, 28(1), 49-55. [CrossRef]
32. 32. Twilley, D., Lall, N. (2018). The role of natural products from plants in the development of anticancer agents. In: Mandal, S.C., Mandal. V., Konishi, T. (Eds.), Natural Products and Drug Discovery, (pp. 139-178). Elsevier. [CrossRef]
33. 33. Sırmatel, F., Duygu, F., Çelik, H., Selek, Ş., Sırmatel, Ö., Gürsoy, B., Eriş, F.N. (2009). Kronik Viral Hepatit Olgularında Total Oksidatif Seviye ve Total Antioksidan Kapasitenin Değerlendirilmesi. Klinik Journal/Klimik Dergisi, 22(3), 92-96.
34. 34. Djeridane, A., Yousfi, M., Nadjemi, B., Boutassouna, D., Stocker, P.,Vidal, N. (2006). Antioxidant activity of some Algerian medicinal plants extracts containing phenolic compounds. Food Chemistry, 97(4), 654-660. [CrossRef]
35. 35. Oliveira, A.M.F., Pinheiro, L.S., Pereira, C.K.S., Matias, W.N., Gomes, R.A., Chaves, O.S., Assis, T.S. (2012). Total phenolic content and antioxidant activity of some Malvaceae family species. Antioxidants, 1(1), 33-43. [CrossRef]
36. 36. Muchuweti, M., Nyamukonda, L., Chagonda, L.S., Ndhlala, A.R., Mupure, C., Benhura, M. (2006). Total phenolic content and antioxidant activity in selected medicinal plants of Zimbabwe. International Journal of Food Science & Technology, 41, 33-38. [CrossRef]
37. 37. Kaur, S., Mondal, P. (2014). Study of total phenolic and flavonoid content, antioxidant activity and antimicrobial properties of medicinal plants. Journal of Microbiology & Expermientation, 1(1), 23-28. [CrossRef]
38. 38. Sevindik, M., Akgul, H., Pehlivan, M., Selamoglu, Z. (2017). Determination of therapeutic potential of Mentha longifolia ssp. longifolia. Fresenius Environmental Bulletin, 26(7), 4757-4763.
39. 39. Pehlivan, M., Sevindik, M. (2018). Antioxidant and antimicrobial activities of Salvia multicaulis. Turkish Journal of Agriculture-Food Science and Technology, 6(5), 628-631. [CrossRef]
40. 40. Mohammed, F.S., Akgül, H., Sevindik, M., Khaled, B.M.T. (2018). Phenolic content and biological activities of Rhus coriaria var. zebaria. Fresenius Environmental Bulletin, 27(8), 5694-5702.
41. 41. Mohammed, F.S., Karakaş, M., Akgül, H., Sevindik, M. (2019). Medicinal properties of Allium calocephalum collected from Gara Mountain (Iraq). Fresenius Environmental Bulletin, 28(10), 7419-7426.
42. 42. Mohammed, F.S., Pehlivan, M., Sevindik, M. (2019). Antioxidant, antibacterial and antifungal activities of different extracts of Silybum marianum collected from Duhok (Iraq). International Journal of Secondary Metabolite, 6(4), 317-322. [CrossRef]
43. 43. Mohammed, F.S., Şabik, A.E., Sevindik, E., Pehlivan, M., Sevindik, M. (2020). Determination of Antioxidant and Oxidant Potentials of Thymbra spicata Collected from Duhok-Iraq. Turkish Journal of Agriculture-Food Science and Technology, 8(5), 1171-1173. [CrossRef]
44. 44. Chen, Y., Tseng, S.H., Lai, H.S., Chen, W. J. (2004). Resveratrol-induced cellular apoptosis and cell cycle arrest in neuroblastoma cells and antitumor effects on neuroblastoma in mice. Surgery, 136(1), 57-66. [CrossRef]
45. 45. Kara, A. (2012). Doktora Tezi. Resveratrolün Meme Kanseri Hücre Kültüründeki P53 Aracılı Apoptoz Aktivasyonunun Ve Sitotoksisitesinin Polimeraz Zincir Reaksiyonu (PCR), İmmunsitokimya ve MTT Yöntemleriyle Araştırılması. Histoloji ve Embriyoloji Ana Bilim Dalı, Sağlık Bilimleri Enstitüsü, Atatürk Üniversitesi, Erzurum, Türkiye.
46. 46. Arat, E. (2012). Yüksek LisansTezi. Alchemilla L. Cinsine Ait Bazı Türlerden Elde Edilen Ekstrelerin MCF-7 Kanser ve L929 Fibroblast Hücrelerine Etkisi. Biyoloji Anabilim Dalı, Fen Bilimleri Enstitüsü, Kırıkkale Üniversitesi, Kırıkkale, Türkiye.
47. 47. Duman, İ. (2018). Yüksek LisansTezi. Endometriyum Adenokarsinoma HEC1A Hücrelerinde D Vitamininin Proliferasyon, Migrasyon ve İnvazyon Üzerine Etkisi. Tıbbi Farmakoloji Anabilim Dalı. Sağlık Bilimleri Enstitüsü, Mersin Üniversitesi, Mersin, Türkiye.
48. 48. Pereira, L.O.M., Vilegas, W., Tangerina, M.M.P., Arunachalam, K., Balogun, S.O., Orlandi-Mattos, P. E., Oliveira Martins, D.T. (2018). Lafoensia pacari A. St.-Hil.: Wound healing activity and mechanism of action of standardized hydroethanolic leaves extract. Journal of Ethnopharmacology, 219, 337-350. [CrossRef]