Sub-cytotoxic concentrations of ionic silver promote the proliferation of human keratinocytes by inducing the production of reactive oxygen species

doi:10.1007/s11684-017-0550-7

Front. Med.

2018, Vol. 12

Issue (3) : 289-300 https://doi.org/10.1007/s11684-017-0550-7

RESEARCH ARTICLE

Sub-cytotoxic concentrations of ionic silver promote the proliferation of human keratinocytes by inducing the production of reactive oxygen species

Xiaodong Duan^1,², Daizhi Peng^1,³(

), Yilan Zhang¹, Yalan Huang¹, Xiao Liu¹, Ruifu Li¹, Xin Zhou¹, Jing Liu¹

¹. Institute of Burn Research, Southwest Hospital, Third Military Medical University, Chongqing 400038, China
². Burn and Plastic Surgery Department, 209 Hospital of PLA, Mudanjiang 157011, China
³. Tissue Engineering Research Unit, State Key Laboratory of Trauma, Burns and Combined Injury, Third Military Medical University, Chongqing 400038, China

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Abstract

Silver-containing preparations are widely used in the management of skin wounds, but the effects of silver ions on skin wound healing remain poorly understood. This study investigated the effects of silver ions (Ag⁺) on the proliferation of human skin keratinocytes (HaCaT) and the production of intracellular reactive oxygen species (ROS). After treating HaCaT cells with Ag⁺ and/or the active oxygen scavenger N-acetyl cysteine (NAC), cell proliferation and intracellular ROS generation were assessed using CCK-8 reagent and DCFH-DA fluorescent probe, respectively. In addition, 5-bromo-2-deoxyUridine (BrdU) incorporation assays, cell cycle flow cytometry, and proliferating cell nuclear antigen (PCNA) immunocytochemistry were conducted to further evaluate the effects of sub-cytotoxic Ag⁺ concentrations on HaCaT cells. The proliferation of HaCaT cells was promoted in the presence of 10⁻⁶ and 10⁻⁵ mol/L Ag⁺ at 24, 48, and 72 h. Intracellular ROS generation also significantly increased for 5–60 min after exposure to Ag⁺. The number of BrdU-positive cells and the presence of PCNA in HaCaT cells increased 48 h after the addition of 10⁻⁶ and 10⁻⁵ mol/L Ag⁺, with 10⁻⁵ mol/L Ag⁺ markedly increasing the cell proliferation index. These effects of sub-cytotoxic Ag⁺ concentrations were repressed by 5 mmol/L NAC. Our results suggest that sub-cytotoxic Ag⁺ concentrations promote the proliferation of human keratinocytes and might be associated with a moderate increase in intracellular ROS levels. This study provides important experimental evidence for developing novel silver-based wound agents or dressings with few or no cytotoxicity.

Keywords ionic silver human keratinocyte cell proliferation reactive oxygen species active oxygen scavenger NAC

Corresponding Author(s): Daizhi Peng

Just Accepted Date: 04 August 2017 Online First Date: 03 November 2017 Issue Date: 04 May 2018

Cite this article:

Xiaodong Duan,Daizhi Peng,Yilan Zhang, et al. Sub-cytotoxic concentrations of ionic silver promote the proliferation of human keratinocytes by inducing the production of reactive oxygen species[J]. Front. Med., 2018, 12(3): 289-300.

URL:

https://academic.hep.com.cn/fmd/EN/10.1007/s11684-017-0550-7
https://academic.hep.com.cn/fmd/EN/Y2018/V12/I3/289

Fig.1 Ag⁺ influences the proliferation of HaCaT cells. (A–E) Relative proliferation ratios of HaCaT cells following treatment with various concentrations of Ag⁺. *P<0.05, **P<0.01, experimental versus negative control groups. (F) HaCaT cells (40× and 200×magnifications) following treatment with Ag⁺ for 48 h.

Fig.2 Effects of Ag⁺ on ROS generation in HaCaT cells. (A–F) Relative fluorescence intensity of DCFH-DA in HaCaT cells following treatment with Ag⁺. *P<0.05, **P<0.01 experimental versus negative control groups. (G) Representative images of HaCaT cells (400× magnification) after treatment with Ag⁺ for 48 h.

Fig.3 Effects of NAC on the proliferation of HaCaT cells treated with Ag⁺.

Fig.4 Effects of NAC and silver ion on ROS production in HaCaT cells. (A–F) Relative fluorescence intensity of intracellular DCFH-DA in HaCaT cells after exposure to Ag⁺ and NAC (n = 3). (G) Representative images of intracellular DCFH-DA 60 min after HaCaT cells were treated with Ag⁺ and NAC (400× magnification).

Fig.5 Effects of NAC and Ag⁺ on BrdU incorporation and PCNA expression in HaCaT cells. (A) Percentage of BrdU-positive cells reflecting the level of cell proliferation. (B) Immunofluorescent staining of PCNA. (C) Representative images (400× magnification) showing BrdU incorporation (red) and the presence of PCNA (green). Cell nuclei were stained with DAPI (blue).

Tab.1 Effects of NAC and Ag⁺ on cell cycle in HaCaT cells

Fig.6 Cell cycle distribution 48 h after treatment with Ag⁺ and NAC treatment. (A) 0 mol/L Ag⁺. (B) 10⁻⁵ mol/L Ag⁺. (C) 10⁻⁵ mol/L Ag⁺ and 5 mmol/L NAC.

1	Moyer CA, Brentano L, Gravens DL, Margraf HW Jr, Monafo WW Jr. Treatment of large human burns with 0.5 per cent silver nitrate solution. Arch Surg 1965; 90(6): 812–867 https://doi.org/10.1001/archsurg.1965.01320120014002 pmid: 14333527
2	Fox CL Jr. Silver sulfadiazine—a new topical therapy for Pseudomonas in burns. Therapy of Pseudomonas infection in burns. Arch Surg 1968; 96(2): 184–188 https://doi.org/10.1001/archsurg.1968.01330200022004 pmid: 5638080
3	Chopra I. The increasing use of silver-based products as antimicrobial agents: a useful development or a cause for concern? J Antimicrob Chemother 2007; 59(4): 587–590 https://doi.org/10.1093/jac/dkm006 pmid: 17307768
4	Swathy JR, Sankar MU, Chaudhary A, Aigal S, Anshup T, Pradeep. Antimicrobial silver: an unprecedented anion effect. Sci Rep 2014; 4: 7161 https://doi.org/10.1038/srep07161 pmid: 25418185
5	Mijnendonckx K, Leys N, Mahillon J, Silver S, Van Houdt R. Antimicrobial silver: uses, toxicity and potential for resistance. Biometals 2013; 26(4): 609–621 https://doi.org/10.1007/s10534-013-9645-z pmid: 23771576
6	Lansdown A, Williams A. Bacterial resistance to silver-based antibiotics. Nurs Times 2007; 103(9): 48–49 pmid: 17375724
7	Burd A, Kwok CH, Hung SC, Chan HS, Gu H, Lam WK, Huang L. A comparative study of the cytotoxicity of silver-based dressings in monolayer cell, tissue explant, and animal models. Wound Repair Regen 2007; 15(1): 94–104 https://doi.org/10.1111/j.1524-475X.2006.00190.x pmid: 17244325
8	Berger TJ, Spadaro JA, Chapin SE, Becker RO. Electrically generated silver ions: quantitative effects on bacterial and mammalian cells. Antimicrob Agents Chemother 1976; 9(2): 357–358 https://doi.org/10.1128/AAC.9.2.357 pmid: 944551
9	Arora S, Jain J, Rajwade JM, Paknikar KM. Interactions of silver nanoparticles with primary mouse fibroblasts and liver cells. Toxicol Appl Pharmacol 2009; 236(3): 310–318 https://doi.org/10.1016/j.taap.2009.02.020 pmid: 19269301
10	Zanette C, Pelin M, Crosera M, Adami G, Bovenzi M, Larese FF, Florio C. Silver nanoparticles exert a long-lasting antiproliferative effect on human keratinocyte HaCaT cell line. Toxicol In Vitro 2011; 25(5): 1053–1060 https://doi.org/10.1016/j.tiv.2011.04.005 pmid: 21501681
11	Poon VKM, Burd A. In vitro cytotoxity of silver: implication for clinical wound care. Burns 2004; 30(2): 140–147 https://doi.org/10.1016/j.burns.2003.09.030 pmid: 15019121
12	Morones-Ramirez JR, Winkler JA, Spina CS, Collins JJ. Silver enhances antibiotic activity against gram-negative bacteria. Sci Transl Med 2013; 5(190): 190ra81 https://doi.org/10.1126/scitranslmed.3006276 pmid: 23785037
13	Thomas GW, Rael LT, Bar-Or R, Shimonkevitz R, Mains CW, Slone DS, Craun ML, Bar-Or D. Mechanisms of delayed wound healing by commonly used antiseptics. J Trauma 2009, 66:82–90, discussion 90–91 https://doi.org/10.1097/TA.0b013e31818b146d
14	Michaels JA, Campbell B, King B, Palfreyman SJ, Shackley P, Stevenson M. Randomized controlled trial and cost-effectiveness analysis of silver-donating antimicrobial dressings for venous leg ulcers (VULCAN trial). Br J Surg 2009; 96(10): 1147–1156 https://doi.org/10.1002/bjs.6786 pmid: 19787753
15	White R, Cutting K, Ousey K, Butcher M, Gray D, Flanagan M, Donnelly J, McIntosh C, Kingsley A, Fletcher J, Chadwick P, Gethin G, Beldon P. Randomized controlled trial and cost-effectiveness analysis of silver-donating antimicrobial dressings for venous leg ulcers (VULCAN trial) (Br J Surg 2009; 96: 1147-1156). Br J Surg 2010; 97(3): 459–460 https://doi.org/10.1002/bjs.7017 pmid: 20140943
16	Vermeulen H, van Hattem JM, Storm-Versloot MN, Ubbink DT. Topical silver for treating infected wounds. Cochrane Database Syst Rev 2007; (1): CD005486 pmid: 17253557
17	Storm-Versloot MN, Vos CG, Ubbink DT, Vermeulen H. Topical silver for preventing wound infection. Cochrane Database Syst Rev 2010; (3): CD006478 pmid: 20238345
18	Aziz Z, Abu SF, Chong NJ. A systematic review of silver-containing dressings and topical silver agents (used with dressings) for burn wounds. Burns 2012; 38(3): 307–318 https://doi.org/10.1016/j.burns.2011.09.020 pmid: 22030441
19	Gordon O, Vig Slenters T, Brunetto PS, Villaruz AE, Sturdevant DE, Otto M, Landmann R, Fromm KM. Silver coordination polymers for prevention of implant infection: thiol interaction, impact on respiratory chain enzymes, and hydroxyl radical induction. Antimicrob Agents Chemother 2010; 54(10): 4208– 4218 https://doi.org/10.1128/AAC.01830-09 pmid: 20660682
20	Foldbjerg R, Olesen P, Hougaard M, Dang DA, Hoffmann HJ, Autrup H. PVP-coated silver nanoparticles and silver ions induce reactive oxygen species, apoptosis and necrosis in THP-1 monocytes. Toxicol Lett 2009; 190(2): 156–162 https://doi.org/10.1016/j.toxlet.2009.07.009 pmid: 19607894
21	Miyayama T, Arai Y, Suzuki N, Hirano S. Mitochondrial electron transport is inhibited by disappearance of metallothionein in human bronchial epithelial cells following exposure to silver nitrate. Toxicology 2013; 305: 20–29 https://doi.org/10.1016/j.tox.2013.01.004 pmid: 23333424
22	Allen RG, Tresini M. Oxidative stress and gene regulation. Free Radic Biol Med 2000; 28(3): 463–499 https://doi.org/10.1016/S0891-5849(99)00242-7 pmid: 10699758
23	Yamamoto A, Honma R, Tanaka A, Sumita M. Generic tendency of metal salt cytotoxicity for six cell lines. J Biomed Mater Res 1999; 47(3): 396–403 https://doi.org/10.1002/(SICI)1097-4636(19991205)47:3<396::AID-JBM15>3.0.CO;2-R pmid: 10487892
24	Martin KR, Barrett JC. Reactive oxygen species as double-edged swords in cellular processes: low-dose cell signaling versus high-dose toxicity. Hum Exp Toxicol 2002; 21(2): 71–75 https://doi.org/10.1191/0960327102ht213oa pmid: 12102499
25	Sena LA, Chandel NS. Physiological roles of mitochondrial reactive oxygen species. Mol Cell 2012; 48(2): 158–167 https://doi.org/10.1016/j.molcel.2012.09.025 pmid: 23102266
26	Sauer H, Wartenberg M, Hescheler J. Reactive oxygen species as intracellular messengers during cell growth and differentiation. Cell Physiol Biochem 2001; 11(4): 173–186 https://doi.org/10.1159/000047804 pmid: 11509825
27	Samuni Y, Goldstein S, Dean OM, Berk M. The chemistry and biological activities of N-acetylcysteine. Biochim Biophys Acta. 2013;1830(8): 4117–4129 https://doi.org/10.1016/j.bbagen.2013.04.016
28	Bateman DN, Dear JW, Thanacoody HK, Thomas SH, Eddleston M, Sandilands EA, Coyle J, Cooper JG, Rodriguez A, Butcher I, Lewis SC, Vliegenthart AD, Veiraiah A, Webb DJ, Gray A. Reduction of adverse effects from intravenous acetylcysteine treatment for paracetamol poisoning: a randomised controlled trial. Lancet 2014; 383(9918): 697–704 https://doi.org/10.1016/S0140-6736(13)62062-0 pmid: 24290406
29	Tominaga H, Ishiyama M, Ohseto F, Sasamoto K, Hamamoto T, Suzuki K, Watanabe M. A water-soluble tetrazolium salt useful for colorimetric cell viability assay. Anal Commun 1999; 36(2): 47–50 https://doi.org/10.1039/a809656b
30	Bosq J, Bourhis J. Bromodeoxyuridine (BrdU). Analysis of cellular proliferation. Ann Pathol 1997; 17(3): 171–178 (in French) pmid: 9296576
31	Chen AC, Arany PR, Huang YY, Tomkinson EM, Sharma SK, Kharkwal GB, Saleem T, Mooney D, Yull FE, Blackwell TS, Hamblin MR. Low-level laser therapy activates NF-κB via generation of reactive oxygen species in mouse embryonic fibroblasts. PLoS One 2011; 6(7): e22453 https://doi.org/10.1371/journal.pone.0022453 pmid: 21814580
32	Burdon RH. Superoxide and hydrogen peroxide in relation to mammalian cell proliferation. Free Radic Biol Med 1995; 18(4): 775–794 https://doi.org/10.1016/0891-5849(94)00198-S pmid: 7750801
33	Ruiz-Ginés JA, López-Ongil S, González-Rubio M, González-Santiago L, Rodrµguez-Puyol M, Rodrµguez-Puyol D. Reactive oxygen species induce proliferation of bovine aortic endothelial cells. J Cardiovasc Pharmacol 2000; 35(1): 109–113 https://doi.org/10.1097/00005344-200001000-00014 pmid: 10630740
34	Egelkrout EM, Mariconti L, Settlage SB, Cella R, Robertson D, Hanley-Bowdoin L. Two E2F elements regulate the proliferating cell nuclear antigen promoter differently during leaf development. Plant Cell 2002; 14(12): 3225–3236 https://doi.org/10.1105/tpc.006403 pmid: 12468739
35	Koundrioukoff S, Jónsson ZO, Hasan S, de Jong RN, van der Vliet PC, Hottiger MO, Hübscher U. A direct interaction between proliferating cell nuclear antigen (PCNA) and Cdk2 targets PCNA-interacting proteins for phosphorylation. J Biol Chem 2000; 275(30): 22882–22887 https://doi.org/10.1074/jbc.M001850200 pmid: 10930425

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