Effects of Uridine and Uridine Nucleotides on Proliferation and Migration of L929 Murine Fibroblast Cell Line

Abstract

The present study aimed to investigate the effects of uridine and the uridine nucleotides uridine-5’-monophosphate (UMP), uridine-5’-diphosphate (UDP) and uridine-5’-triphosphate (UTP) at different concentrations (1, 10 and 100 μM) on cell viability and migration capacity using the L929 murine fibroblast cell line. To assess cytotoxicity and cellular proliferation, the MTT [(3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide)] assay was performed at 24, 48 and 72 hours of incubation, providing a quantitative evaluation of metabolic activity and cell survival. Additionally, the scratch assay was employed and followed up to 96 hours to analyze fibroblast migration, offering insights into the role of these pyrimidines in wound healing and tissue repair. The MTT assay revealed that the highest concentration (100 μM) of UMP or UDP significantly enhanced proliferation of cells at 72 hours while uridine at 10 and 100 μM and UTP at all concentrations tested provided the same effect. In scratch assay UMP or UDP did not cause any significant cell migration while uridine and UTP, at all concentrations, significantly enhanced migration of fibroblast cells at 96 hours. The results demonstrated distinct effects of uridine and its nucleotides on cell viability and migration, with significant benefit in terms of wound healing provided by uridine and UTP, highlighting their potential biological significance and therapeutic implications in regenerative medicine and tissue engineering.

Keywords

• uridine
• L929 murine fibroblast cells
• MTT
• proliferation
• migration
• wound healing

Uridin ve Uridin Nükleotidlerinin L929 Fare Fibroblastik Hücrelerinin Proliferasyonu ve Göçü Üzerindeki Etkileri

Abstract

Bu çalışmanın amacı, üridin ve üridin nükleotidleri üridin-5’-monofosfat (UMP), üridin-5’-difosfat (UDP) ve üridin-5’-trifosfat (UTP)’ın farklı konsantrasyonlarda (1, 10 ve 100 μM) uygulanmasının L929 kemirgen fibroblast hücre kültüründe hücre canlılığı ve göç etme kapasitesine etkilerini incelemektir. Sitotoksisite ve hücresel proliferasyonu değerlendirmek için 24, 48 ve 72 saatlik inkübasyonların sonunda metabolik aktivite ve hücre canlılığı konusunda kantitatif değerlendirme yapılmasını sağlayan MTT [3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazolyum bromür] testi uygulandı. Ayrıca bu pirimidin bileşiklerinin yara iyileşmesi ve doku tamiri konusundaki etkilerini incelemek üzere çizik testi yapılarak fibroblast göçü 96 saate kadar takip edildi. MTT testi ile 72 saat inkübasyon sonunda UMP veya UDP’nin en yüksek (100 μM) konsantrasyonda hücre proliferasyonunu artırırken üridin’in 10 ve 100 μM konsantrasyonlarda ve UTP’nin denenen tüm konsantrasyonlarında aynı etkiyi gösterdiği tespit edildi. Çizik testinde UMP veya UDP anlamlı hücre göçüne neden olmazken 96 saat inkübasyon sonrası üridin ve UTP denenen tüm konsantrasyonlarında hücre göçünü anlamlı olarak artırdı. Bu sonuçlar üridin ve üridin nükleotidlerinin hücre proliferasyonu ve göçü üzerine farklı etkileri olduğunu ve yara iyileşmesi bakımından üridin ve UTP’nin anlamlı faydaları olabileceğini göstermekle birlikte rejeneratif tıp ve doku mühendisliği alanlarındaki potansiyel biyolojik önemlerine ve terapötik kullanımlarına işaret etmektedir.

Keywords

• üridin
• L929 kemirgen fibroblast hücreleri
• MTT
• proliferasyon
• migrasyon
• yara iyileşmesi

References

1. 1. Lane AN, Fan TWM. Regulation of mammalian nucleotide metabolism and biosynthesis. Nucleic Acids Res. 2015;43(4):2466–2485. doi:10.1093/nar/gkv047
2. 2. Traut TW. Physiological concentrations of purines and pyrimidines. Mol Cell Biochem. 1994;140(1):1–22. doi:10.1007/BF00928361
3. 3. Lecca D, Ceruti S. Uracil nucleotides: From metabolic intermediates to neuroprotection and neuroinflammation. Biochem Pharmacol. 2008;75(10):1869–1881. doi:10.1016/j.bcp.2007.12.009
4. 4. Koyuncuoglu T, Turkyilmaz M, Goren B, Cetinkaya M, Cansev M, Alkan T. Uridine protects against hypoxic-ischemic brain injury by reducing histone deacetylase activity in neonatal rats. Restor Neurol Neurosci. 2015;33(5):777–784. doi:10.3233/RNN-150549
5. 5. Cicko S, Grimm M, Ayata K, vd. Uridine supplementation exerts anti-inflammatory and anti-fibrotic effects in an animal model of pulmonary fibrosis. Respir Res. 2015;16(1):105. doi:10.1186/s12931-015-0264-9
6. 6. Al, Nevin; Cakir, Aysen; Koc, Cansu; Cansev, Mehmet; Alkan T. Antioxidative effects of uridine in a neonatal rat model of hyperoxic brain injury. TURKISH J Med Sci. 2020;50(8):2059–2066. doi:10.3906/sag-2002-14
7. 7. Cakir A, Ocalan Esmerce B, Aydin B, vd. Effects of uridine administration on hippocampal matrix metalloproteinases and their endogenous inhibitors in REM sleep-deprived rats. Brain Res. 2022;1793:148039. doi:10.1016/j.brainres.2022.148039
8. 8. Liu Z, Li W, Geng L, vd. Cross-species metabolomic analysis identifies uridine as a potent regeneration promoting factor. Cell Discov. 2022;8(1):6. doi:10.1038/s41421-021-00361-3

9. 9. Plikus M V., Wang X, Sinha S, vd. Fibroblasts: Origins, definitions, and functions in health and disease. Cell. 2021;184(15):3852–3872. doi:10.1016/j.cell.2021.06.024
10. 10. Viennet C, Muret P. Fibroblast Evaluation: Extracellular Matrix Synthesis. Içinde: Measuring the Skin. ; 2015:1–5. doi:10.1007/978-3-319-26594-0_124-1
11. 11. Wong ZY, Nee E, Coles M, Buckley CD. Why does understanding the biology of fibroblasts in immunity really matter? PLoS Biol. 2023;21(2). doi:10.1371/journal.pbio.3001954
12. 12. Gu M, Xu J, Han C, vd. Effects of Berberine on Cell Cycle, DNA, Reactive Oxygen Species, and Apoptosis in L929 Murine Fibroblast Cells. Evidence-based Complement Altern Med. 2015;2015:796306. doi:10.1155/2015/796306
13. 13. Mishra SK, Braun N, Shukla V, vd. Extracelluar nucleotide signaling in adult neural stem cells: Synergism with growth factor-mediated cellular proliferation. Development. 2006;133(4):675–684. doi:10.1242/dev.02233
14. 14. Milosevic J, Brandt A, Roemuss U, vd. Uracil nucleotides stimulate human neural precursor cell proliferation and dopaminergic differentiation: Involvement of MEK/ERK signalling. J Neurochem. 2006;99(3):913–923. doi:10.1111/j.1471-4159.2006.04132.x
15. 15. Braun OÖ, Lu D, Aroonsakool N, Insel PA. Uridine triphosphate (UTP) induces profibrotic responses in cardiac fibroblasts by activation of P2Y2 receptors. J Mol Cell Cardiol. 2010;49(3):362–369. doi:10.1016/j.yjmcc.2010.05.001
16. 16. Mergen HS, Duzyer Gebizli S, Ermis E, Cansev M, Isik Dokuzoglu S, Goktalay G. Uridine-Loaded Polycaprolactone Nanofiber Mats as a Novel Wound Dressing. Fibers Polym. 2024;25(7):2471–2484. doi:10.1007/s12221-024-00615-3
17. 17. Khezri MK, Turkkan A, Koc C, vd. Uridine Treatment Improves Nerve Regeneration and Functional Recovery in a Rat Model of Sciatic Nerve Injury. Turk Neurosurg. 2022;32(6):935–943. doi:10.5137/1019-5149.JTN.36142-21.2
18. 18. Karimi Khezri M, Turkkan A, Koc C, vd. Anti-apoptotic and anti-oxidant effects of systemic uridine treatment in an experimental model of sciatic nerve injury. Turk Neurosurg. 2021;31(3):373–378. doi:10.5137/1019-5149.jtn.31127-20.3
19. 19. Ozmarasali AI, Koc C, Huseyin U, Mehmet C, Ilker Mustafa K, Ahmet B. Mediation of epigenetic mechanisms in the regenerative effect of uridine in a rat model of sciatic nerve injury. Turk Neurosurg. 2023;34(6):1122–1132. doi:10.5137/1019-5149.jtn.45425-23.2
20. 20. Almadani YH, Vorstenbosch J, Davison PG, Murphy AM. Wound Healing: A Comprehensive Review. Semin Plast Surg. 2021;35(3):141–144. doi:10.1055/s-0041-1731791
21. 21. Landén NX, Li D, Ståhle M. Transition from inflammation to proliferation: a critical step during wound healing. Cell Mol Life Sci. 2016;73(20):3861–3885. doi:10.1007/s00018-016-2268-0
22. 22. Jacobson KA, Delicado EG, Gachet C, vd. Update of P2Y receptor pharmacology: IUPHAR Review 27. Br J Pharmacol. 2020;177(11):2413–2433. doi:10.1111/bph.15005