Investigation of the Effect of Hydrogen Sulfide Donor Sodium Hydrogen Sulfide on In Vitro Wound Healing

Öz

In the wound healing process, various biological activities such as cell proliferation, cell differentiation, and cell death are mediated by growth factors. Gasotransmitters are signaling molecules that have been shown to be effective in the wound healing process. H2S, a member of the gasotransmitter family, is reported to function as a regulatory molecule in wound healing in in vivo studies. The indirect effect of H2S in the wound healing process is observed through the activation of various signaling pathways. This study investigates whether H2S has a direct effect rather than an indirect effect. In this context, the impact of NaHS, an H2S donor, on the expression levels of various genes involved in in vitro wound healing has been evaluated. Immortalized human keratinocyte cells were incubated with NaHS at concentrations of 50 μg, 25 μg, 10 μg, 5 μg, and 1μg for 24, 48, and 72 hours and cell viability was determined by MTS analysis. Changes in the expression of TGF-β1, TGF-β3, VEGF and K17 genes were determined using qRT-PCR, and the data were calculated using the ΔΔCT method. In terms of cell viability, it was determined that the 1μg dose of NaHS is a non-toxic dose. While NaHS application significantly increased K17 mRNA expression, it didn’t cause significant changes in TGF-β1, TGF-β3 and VEGF expression. It was concluded that NaHS doesn’t have a direct effect on in vitro wound healing.

Anahtar Kelimeler

• H2S
• NaHS
• wound healing

Hidrojen Sulfid Donörü Sodyum Hidrojen Sülfür’ün In Vitro Yara İyileşmesi Üzerine Etkisinin Araştırılması

Öz

Yara iyileşmesi sürecinde büyüme faktörlerinin hücre proliferasyonu, hücre farklılaşması ve hücre ölümü gibi çeşitli biyolojik aktiviteleri mevcuttur. Gazotransmitterler yara iyileşme sürecinde etkinliği gösterilmiş sinyal molekülleridir. Gazotransmitter ailesinin bir üyesi olan H2S’in in vivo çalışmalarda yara iyileşmesinde düzenleyici bir molekül olarak görev aldığı bildirilmektedir. H2S’in yara iyileşme sürecinde çeşitli sinyal yolaklarının aktivasyonu aracılığıyla indirekt etkisinin olduğu görülmektedir. Bu çalışmada H2S’in indirekt etkisinden ziyade direkt etkisinin olup olmadığı araştırılmıştır. Bu bağlamda H2S donörü olan NaHS’in in vitro yara iyileşmesinde fonksiyon gösteren çeşitli genlerin ekspresyon düzeylerine olan etkisi değerlendirilmiştir. İmmortalize insan keratinosit hücreleri 50μg, 25μg, 10μg, 5μg, 1μg konsantrasyonda NaHS ile 24, 48 ve 72 saat boyunca inkübe edilmiş ve MTS analizi ile hücre canlığı belirlenmiştir. TGF-β1, TGF-β3, VEGF ve K17 gen ifadelerindeki değişimler qRT-PCR yöntemiyle belirlenerek elde edilen veriler ΔΔCT metodu ile hesaplanmıştır. Hücre canlılığı açısından farklı konsantrasyonlarda uygulanan NaHS’in 1μg’lık dozunun toksik olmayan doz olduğu belirlenmiştir. NaHS uygulamasının K17 mRNA ekspresyonunu anlamlı düzeyde arttırırken TGF-β1, TGF-β3 ve VEGF ekspresyonunda anlamlı değişikliğe yol açmadığı saptanmıştır. NaHS’in in vitro yara iyileşmesi üzerine direkt etkisinin olmadığı sonucuna varılmıştır.

Anahtar Kelimeler

• H2S
• NaHS
• yara iyileşmesi

Etik Beyan

Çalışmamızda ticari olarak satın alınan insan hücre hattı kullanılmıştır. Etik kurul onayına gerek yoktur.

Kaynakça

1. 1. Velnar T, Bailey T, Smrkolj V, J. Int. Med. Res. 2009, 37, 1528–1542.
2. 2. Broughton G.I.I, Janis J.E, Attinger C.E, Plast. Reconstr. Surg. 2006,117.
3. 3. Shibuya N, Mikami Y, Kimura Y, Nagahara N. & Kimura H. Vascular endothelium expresses 3-mercaptopyruvate sulfurtransferase and produces hydrogen sulfide. J. Biochem 2009;146, 623–626.
4. 4. Li L, Rose P and Moore P. K. Hydrogen sulfide and cell signaling, Annu. Rev. Pharmacol. Toxicol., 2011;51,169-167.
5. 5. S.A. Coavoy-Sánchez, S.K. Costa, M.N. Muscará, Hydrogen sulfide and dermatological diseases, Br. J. Pharmacol. 2019;177 (4),857–865.
6. 6. R.M. Osipov, M.P. Robich, J. Feng, Y. et. al.Effect of hydrogen sulfide in a porcine model of myo- cardial ischemia- reperfusion: comparison of different administration regimens and characterization of the cellular mechanisms of protection, J. Cardiovasc. Pharmacol. 2009;54 (4),287–297.
7. 7. Wallace JL, Dicay M, McKnight W, Martin GR. Hydrogen sulfide enhances ulcer healing in rats. FASEB J 2007;21:4070–4076
8. 8. Papapetropoulos A, Pyriochou A, Altaany Z, et al. Hydrogen sulfide is an endogenous stimulator of angiogenesis. Proc Natl Acad Sci U S A 2009;106: 21972–21977.

9. 9. Asai J, Takenaka H, Hirakawa S, et al. Topical Simvastatin Ac- celerates Wound Healing in Diabetes by Enhancing Angiogenesis and Lymphangiogenesis. Am J Pathol 2012;181:2217- 24.
10. 10. Liu X, Han X, Shang Y, et. al. Hydrogen sulfide releasing poly(γ-glutamic acid) biocomposite hydrogel with monitoring, antioxidant, and antibacterial properties for diabetic wound healing. International Journal of Biological Macromolecules 253 (2023) 127053.
11. 11. Zhang Y, Yue T, Gu W, et. al. pH-responsive hierarchical H2S-releasing nano-disinfectant with deep-penetrating and anti-inflammatory properties for synergistically enhanced eradication of bacterial biofilms and wound infection. Journal of Nanobiotechnology (2022) 20:55.
12. 12. Wang F-Z, Zhou H, Wang H-Y. et. al. Hydrogen sulfide prevents arterial medial calcification in rats with diabetic nephropathy. BMC Cardiovasc Disord 2021;21:495
13. 13. Hongfang J, Ying S, Chaoshu T. et.al. Effects of hydrogen sulfide on hypoxic pulmonary vascular structural remodeling Life Sciences 2006;78,1299–1309 .
14. 14. Cai WJ, Wang MJ, Moore PK et. al. The novel proangiogenic effect of hydrogen sulfide is dependent on Akt phosphorylation. Cardiovasc Res 2007;76: 29–40.
15. 15. Wu D, Li M, Tian W. Et. al. Hydrogen sulfide acts as a double-edged sword in human hepatocellular carcinoma cells through EGFR/ ERK/MMP-2 and PTEN/AKT signaling pathways. Sci Rep 2017;7: 5134.
16. 16. Barrientos S, Stojadinovic O, Golinko MS, Brem H, Tomic-Canic M. Growth factors and cytokines in wound healing. Wound Repair and Regeneration. 2008;16(5):585– 601.
17. 17. Zhang X, Yin M and Zhang L-J. Keratin 6, 16 and 17—Critical Barrier Alarmin Molecules in Skin Wounds and Psoriasis. Cells 2019, 8, 807.
18. 18. Mustoe TA, O’Shaughnessy K, Loeters O. Chronic wound pathogenesis and current treatment strategies: a unifying hypothesis. Plast Reconstr Surg 2006;117:35-41.
19. 19. Peplow PV, Chatterjee MP. A review of the influence of growth factors and cytokines in in vitro human keratinocyte migration. Cytokine. 2013;62:1–21.
20. 20. Lichtman MK, Otero-Vinas M, Falanga V. Transforming growth factor beta (TGF-β) isoforms in wound healing and fibrosis. Wound Repair Regen. 2016;24(2):215-22.
21. 21. Shah M, Foreman DM, Ferguson MW. Neutralising antibody to TGF-β1,2 reduces cutaneous scarring in adult rodents. Journal of Cell Science 1994;107:1137-1157.
22. 22. Shah M, Foreman DM, Ferguson MWJ. Neutralisation of TGF-β1 and TGF-β2 or exogenous addition of TGF-β3 to cutaneous rat wounds reduces scarring. Journal of Cell Science 1995;108: 985-1002
23. 23. Le M, Naridze R, Morrison J, Biggs LC, Rhea L, Schutte BC, et al. Transforming growth factor Beta 3 is required for excisional wound repair in vivo. PLoS One 2012; 7: e48040.
24. 24. Li L, Xiao T, Li F. et. al. Hydrogen sulfide reduced renal tissue fibrosis by regulating autophagy in diabetic rats Molecular Medıcıne Reports 2017;16: 1715-1722.
25. 25. Zhang Y, Wang J, Li H. et. al. Hydrogen sulfide suppresses transforming growth factor-β1-induced differentiation of human cardiac fibroblasts into myofibroblasts. Sci China Life Sci November 2015;58,1126-1134.
26. 26.Wang Z, Yin X, Gao Y, et. al. The protective effect ofhydrogen sulfide on systemic sclerosis associated skin and lung fibrosis in mice model. SpringerPlus 2016; 5:1084.
27. 27.Pupo E, Pla AF, Avanzato D. Hydrogen sulfide promotes calcium signals and migration in tumor- derived endothelial cells. Free Radic Biol Med 2011;51: 1765– 1773.
28. 28.Frank S, Stallmeyer B, Kâmpfer H, Kolb N, Pfeilschifter J. Nitric oxide triggers enhanced induction o f vascular endothelial growthfactor expression in cultured keratinocytes(HaCaT) and during cutaneous wound repair. FASEB J1999;13:2002-14.
29. 29.Luo Y, Diao H, Xia S, Dong L, Chen J, Zhang J. Aphysiologically active polysaccharide hydrogel promoteswound healing. J Biomed Material Res A 2010;94:193-204.
30. 30.Karantza V. Keratins in health and cancer: more than mere epithelial cell markers. Oncogene 2011;30(2):127–138.
31. 31.Paladini RD, Takahashi K, Bravo NS, Coulombe PA. Onset ofre-epithelialization after skin injury correlates with a reorganization of keratin filaments in wound edge keratinocytes: defining a potential role for keratin 16. J Cell Biol 1996;132:381–97.
32. 32.Paladini RD, Coulombe PA. Directed expression of keratin 16 to the progenitor basal cells of transgenic mouse skin delays skin maturation. J Cell Biol 1998;142:1035–5
33. 33.Ishikawa K, Sumiyoshi H, Matsuo N. Epiplakin accelerates the lateral organization of keratin filaments during wound healing. Journal of Dermatological Science 60 2010;95–104.
34. 34.Kerns ML, Hakim JM, Lu RG, Guo Y, Berroth A, Kaspar RL,et al. Oxidative stress and dysfunctional NRF2 underlie pachyonychia congenita pheno- types. J Clin Invest2016;126:2356e66.
35. 35.Lessard JC, Coulombe PA. Keratin 16-null mice develop palmoplantar kera- toderma, a hallmark feature of pachyonychiacongenita and related disor- ders. J InvestDermatol 2012;132:1384e91.
36. 36.Calvert J. W, Jha S, Gundewar S, et. al. Hydrogen sulfidemediates cardioprotection through Nrf2 signaling, Circ. Res, 2009;105, 365–374.
37. 37.Vaamonde-García C, Burguera E.F, Vela-Anero A, et. al.Intraarticular Administration Effect of Hydrogen Sulfide on an In Vivo Rat Model of Osteoarthritis. Int. J. Mol. Sci. 2020, 21, 7421.
38. 38.Testaia L, Citia V, Martellia A, Brogia S, Calderone V. Role ofhydrogen sulfide in cardiovascular ageing Pharmacological Research 160 (2020) 105125.