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Frontiers of Medicine

ISSN 2095-0217

ISSN 2095-0225(Online)

CN 11-5983/R

Postal Subscription Code 80-967

2018 Impact Factor: 1.847

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 Duan1,2, Daizhi Peng1,3(), 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 106 and 105 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 106 and 105 mol/L Ag+, with 105 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 Authors: 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:  
http://academic.hep.com.cn/fmd/EN/10.1007/s11684-017-0550-7
http://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).
Ag+ (mol/L)00106106105105
NAC (mmol/L)050505
G1 (%)48.23±5.5047.39±4.9647.49±5.9650.00±8.7039.91±2.23a50.15±6.72b
S (%)47.33±4.5048.42±4.8248.11±5.4145.82±5.3353.46±5.9646.84±6.32
G2 (%)4.44±4.564.20±1.074.41±3.184.19±4.866.64±7.963.01±3.67
PI51.77±5.4952.62±4.9652.52±5.9750.00±8.7060.10±2.23a 49.85±6.71b
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) 105 mol/L Ag+. (C) 105 mol/L Ag+ and 5 mmol/L NAC.
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