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

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

Introduction

In 1965, Moyer et al. [¹] applied 0.5% silver nitrate (AgNO₃) containing 2.94×10⁻² mol/L silver ions (Ag⁺) to large burn wounds. In 1968, Fox [²] used 1% silver sulfadiazine cold cream (2.80×10⁻² mol/L Ag⁺) to treat burn wounds with Pseudomonas infection. Topical agents and wound dressings containing Ag⁺ have since been extensively applied in the field of burn wound treatment, especially given its wide antimicrobial spectrum and low toxicity [³,⁴]. In addition, relatively few strains of bacteria are resistant to the antimicrobial effects of Ag⁺ [⁵,⁶].

Burd et al. [⁷] reported that the concentration of Ag⁺ released from five commonly used surgical dressings ranges from 3 mg/L to 14 mg/L (2.78×10⁻⁵– 1.30× 10⁻⁴ mol/L) and positively correlates with toxicity toward keratinocytes and fibroblasts. According to Berger et al. [⁸] who used 11 representative bacterial strains, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Ag⁺ are 0.03– 1.25 mg/L (2.78×10⁻⁷– 1.16×10⁻⁵ mol/L) and 0.26–10.05 mg/L (2.41×10⁻⁶– 9.32×10⁻⁵ mol/L), respectively. The concentration of Ag⁺ released from these silver dressings is much greater than the MIC and close to the MBC for the 11 strains. High-concentration Ag⁺ in these dressings exerts antimicrobial effects, but its possible cytotoxicity has become a cause of concern [⁹–¹³]. Burd et al. [¹¹] and Collins et al. [¹²] confirmed that the proliferation of human keratinocytes decreases by more than 50% when the concentration of Ag⁺ is 10⁻⁴ mol/L in vitro. Clinically, delays in wound healing following the application of treatments involving Ag⁺ [¹⁴–¹⁸] could be associated with the cytotoxic effects of Ag⁺ [⁷]. To determine the effects of Ag⁺ on cell biology, the underlying mechanisms of sub-cytotoxic Ag⁺ concentrations (<10⁻⁴ mol/L) upon the proliferation of human keratinocytes should be elucidated.

The antibacterial actions of Ag⁺ involve the induction of reactive oxygen species (ROS) generation. In addition, Ag⁺ can damage bacterial DNA by binding with thiol groups, thereby inactivating key enzymes and subsequently affecting physiological processes in bacteria [¹⁹]. Similar mechanisms for Ag⁺ have been observed in mammalian cells [²⁰]. Collins et al. [¹²] clarified that the antibacterial effects of Ag⁺ are related to its role in stimulating ROS generation. Treatment with Ag⁺ at concentrations of 3×10⁻⁵ and 6×10⁻⁵ mol/L increases the levels of intracellular hydroxyl free radicals in Escherichia coli by 2- and 3.57-fold, respectively. Treatment with 1×10⁻⁶ mol/L Ag⁺ for 24 h exerts no obvious effect on the proliferation of human bronchial epithelial cells BEAS-2B but increases the generation of intracellular ROS, particularly hydrogen peroxide (H₂O₂), by 2.4-fold [²¹]. The effects of increased ROS levels on cell proliferation require further study. ROS participate in cell apoptosis and necrosis, and are also involved in signal transduction, transcription factor activation, and gene expression, ultimately promoting cell proliferation and differentiation [²²]. However, little information is available regarding the effect of Ag⁺ on ROS generation in human keratinocytes.

We hypothesized that varying concentrations of Ag⁺ affect the ROS generation and, consequently, proliferation of human keratinocytes. We observed that sub-cytotoxic concentrations of Ag⁺ promoted the proliferation of HaCaT cells and stimulated HaCaT cells to produce appropriate levels of ROS. We used N-acetyl cysteine (NAC) treatment to determine whether clearing ROS could suppress the effects of sub-cytotoxic Ag⁺ concentrations. Our results not only provide new experimental evidence for the role of ROS in the proliferation of human keratinocytes but also shed new light on the development of novel silver-containing agents or dressings with wound healing-promoting effects and few or no cytotoxicity.

Materials and methods

Cell culture

The immortalized HaCaT human skin keratinocyte cell line was cultured in Dulbecco’s modified Eagle’s medium (DMEM, Hyclone, USA) supplemented with 10% fetal bovine serum (Hyclone), 100 U/mL penicillin, and 100 mg/mL streptomycin (Hyclone) in humidified air at 37 °C/5% CO₂. When cultures were 70%–80% confluent, cells were treated with 0.25% trypsin and 0.02 mol/L EDTA for approximately 5 min. Trypsin was inactivated by gently resuspending cells in DMEM. Cell suspensions were centrifuged (200×g, 5 min), supernatants were discarded, and fresh growth medium was added to cell pellets. The number of cells in each suspension was determined, and cells were then seeded on cell culture plates for use in future experiments.

Proliferation assays

HaCaT cells were seeded on 96-well plates (5000 cells/well in 200 µL), with three replicates used for each Ag⁺ concentration. After 5 h, the growth medium was replaced with media containing AgNO₃ (Sigma, USA) at 0 (negative control), 10⁻⁸, 10⁻⁷, 10⁻⁶, 10⁻⁵, or 10⁻⁴ mol/L and then incubated for 24, 48, 72, 96, and 120 h. The cultures were examined after 48 h, and images were acquired using an IX71 inverted microscope (Olympus, Japan). At each time point, 10 µL of Cell Counting Kit-8 (CCK-8) reagent (Dojindo, Japan) was added to each well, and the cultures were incubated at 37 °C for another 4 h. The optical density at 450 nm (OD₄₅₀) was measured using a Varioskan flash microplate reader (Thermo Scientific, USA). Each experiment was repeated five times, with relative cell proliferation activity calculated according to the following formula:

\mathrm{Relative\; cell\; proliferation}(\mathrm{\%})

where OD_et, OD_nc, and OD_bc represent the OD₄₅₀ for the experimental treatment (cells in Ag⁺-supplemented medium), negative control (cells in growth medium lacking Ag⁺), and blank control (growth medium only), respectively.

To assess the effects of NAC on cultures to which Ag⁺ was added, the culture medium was supplemented with 5 mmol/L NAC (Beyotime, China) or with 5 mmol/L NAC and Ag⁺ (10⁻⁶ or 10⁻⁵ mol/L). The cultures were incubated for 48 h at 37 °C, and then 10 µL of CCK-8 reagent was added to each well and allowed to incubate at 37 °C for 4 h. The OD₄₅₀ for each well was determined using a Varioskan flash microplate reader, and proliferation was calculated according to the above formula. Each proliferation assay was independently repeated three times.

Detection of intracellular ROS

HaCaT cells were seeded in 96-well plates (1×10⁴ cells/well in 200 µL), with three replicates used for each Ag⁺ concentration, and then incubated at 37 °C for 24 h. We added 50 µL of 10 µmol/L dichloro-dihydro-fluorescein diacetate (DCFH-DA; Beyotime) to each well and then incubated the plates at 37 °C for 1 h. Culture supernatants were aspirated and discarded, and the cells were rinsed with phosphate-buffered saline (PBS) three times. We then added 200 µL of Ag⁺-supplemented culture medium (10⁻⁸, 10⁻⁷, 10⁻⁶, 10⁻⁵, or 10⁻⁴ mol/L) to wells in each group according to the experimental design. For positive controls, we added 200 µL of 50 µg/mL ROSup (Beyotime) to wells instead of Ag⁺. Culture medium containing DCFH-DA was used as the blank control. Fluorescence intensity was measured at 0, 5, 10, 30, and 60 min after Ag⁺ treatment using a Varioskan flash microplate reader (Thermo Scientific, USA), with excitation and emission at 488 and 525 nm, respectively. Each experiment was repeated five times, and relative fluorescence intensity (RFI) was a ratio of the detected fluorescence signals of the experimental treatment groups to the negative control groups and was calculated according to the following formula:

\mathrm{Relative\; fluorescence\; intensity}(\mathrm{\%})

vwhere F_et, F_nc, and F_bc represent the fluorescence intensity for the experimental treatment (cells in Ag⁺-supplemented medium), negative control (cells in growth medium lacking Ag⁺), and blank control (growth medium containing DCFH-DA) groups, respectively.

To assess the effects of NAC on Ag⁺-induced ROS generation, the cultures were supplemented with 5 mmol/L NAC or with 5 mmol/L NAC and Ag⁺ (10⁻⁶ or 10⁻⁵ mol/L) after HaCaT cells were preloaded with DCFH-DA for 1 h. The RFI was determined 0, 5, 10, 30, and 60 min after the addition of Ag⁺ using a multimode reader. Each assay was independently repeated three times.

Visualization of intracellular ROS and mitochondria using confocal microscopy

HaCaT cells were seeded in 15 mm plates (NEST, China) at a density of 1×10⁴ cells/well in 2 mL of medium, and the plates were incubated at 37 °C. After 24 h, the culture medium was replaced with 500 µL of serum-free DMEM supplemented with 10 µmol/L of DCFH-DA and 1 µmol/L MitoTracker^® Red CMXRos (Molecular Probes, USA) and then incubated at 37 °C for 60 min in the dark. Supernatants were then aspirated, and the cells were rinsed with PBS three times. HaCaT cells were treated with 5 mmol/L NAC or with 5 mmol/L NAC and Ag⁺ (10⁻⁶ or 10⁻⁵ mol/L) for 60 min. The cultures were excited at 488 and 579 nm, and the emitted fluorescence at 525 (ROS, green) and 599 nm (mitochondria, red) was visualized using an LSM780 laser confocal microscope (Carl Zeiss, Germany). The cultures treated with 50 µg/mL ROSup were considered positive controls.

5-bromo-2-deoxyUridine and proliferating cell nuclear antigen immunofluorescence

HaCaT cells were seeded in 24-well plates (1×10⁴ cells/well in 2 mL) containing 15 mm sterile glass coverslips and then incubated at 37 °C. After 5 h, the cells were treated with 5 mmol/L NAC or with 5 mmol/L NAC and Ag⁺ (10⁻⁶ or 10⁻⁵ mol/L) and then incubated at 37 °C for 40 h. We added 10 mmol/L 5-bromo-2-deoxyUridine (BrdU) (BOSTER, China) to each well and then incubated further the cultures at 37 °C for 8 h. After aspirating the supernatants, the cells were rinsed with PBS and then fixed with 4% paraformaldehyde (BOSTER) at room temperature for 10 min. The fixed cells were washed with PBS three times, treated with 0.1% Triton X-100 at room temperature for 10 min, washed three times with PBS, and then exposed to 2 mol/L HCl at 37 °C for 30 min. The cells were again washed with PBS three times and then incubated with immune staining sealing solution (Beyotime) at 37 °C for 1 h. The cells were incubated with polyclonal rabbit anti-human proliferating cell nuclear antigen (PCNA) (1:100 dilution; Proteintech, China) and monoclonal rat anti-BrdU (1:100; BOSTER) antibodies at 4 °C overnight. All coverslips were rinsed with PBS three times, after which the cells were incubated with FITC-labeled polyclonal goat anti-rabbit (1:50; Zhongshan Company, China) and TRITC-labeled polyclonal goat anti-rat (1:50; Golden Bridge) antibodies at 37 °C for 1 h. The cells were rinsed with PBS three times, with 4′,6-diamidino-2-phenylindole (DAPI; Beyotime) added to each well and allowed to incubate at room temperature for 10 min. The cells were rinsed with PBS, and coverslips were mounted with anti-fluorescence quenching fluid (Beyotime). Fluorescence was visualized at 400× magnification under a laser confocal microscope. Experiments were independently repeated three times. The proportion of BrdU-positive cells was calculated from the fields of view at 400× magnification containing≥200 cells according to the following formula:

\mathrm{BrdU}-\mathrm{positive\; cells}(\mathrm{\%})

The fluorescence intensity of PCNA was determined using Image-Pro Express 6.0 (Media Cybernetics, USA).

Cell cycle analysis

Cells were seeded in six-well plates (1×10⁵ cells/well in 5 mL), with two replicates for each treatment. After incubation at 37 °C for 5 h, the cells were treated with 5 mmol/L NAC or with 5 mmol/L NAC and Ag⁺ (10⁻⁶ or 10⁻⁵ mol/L) for 48 h. The cells were trypsinized, harvested, and then fixed with 1 mL of ice-cold 70% ethanol for 24 h. The fixed cells were washed twice with PBS, incubated with a 0.5 mL solution of propidium iodide (Calbiochem) and RNase A for 30 min, and then subjected to flow cytometry on a BD FACSCalibur^TM (BD Biosciences, USA). Each experiment was independently repeated four times.

Statistical analysis

Each experiment was repeated three or more times. Data are presented as \overline{x}\pm s. All statistical analyses were performed using SPSS 18.0 (SPSS Inc., Chicago, IL, USA). Student’s t-test was used to determine statistical significance. Pearson’s correlation test was used to determine correlation coefficients. A P-value less than 0.05 was considered statistically significant.

Results

Sub-cytotoxic concentrations of Ag⁺ promote HaCaT proliferation

For the negative control group, the proliferation of HaCaT cells increased from 0 h (0.206±0.052) to 48 h (1.228±0.222) and plateaued at 96 h (3.194±0.400) (Fig. 1). Exposure to Ag⁺ at 10⁻⁸ and 10⁻⁷ mol/L exerted no significant effect on HaCaT proliferation (P>0.05 at each time point). For 10⁻⁶ mol/L Ag⁺, the relative proliferations at 24, 48, and 72 h were 106.70%±5.24% (P = 0.046), 103.18%±2.39% (P = 0.041), and 105.43%±3.65% (P = 0.029), respectively. The relative proliferation rates of the cells treated with 10⁻⁵ mol/L Ag⁺ were 118.76%±10.72% (P = 0.017), 114.56%±8.72% (P = 0.020), and 108.14%±3.25% (P = 0.005) at 24, 48, and 72 h, respectively. The level of proliferation at 48 h in the 10⁻⁵ mol/L group (114.56%±8.72%) was significantly higher than that in the 10⁻⁶ mol/L group (103.18%±2.39%; P = 0.041). When Ag⁺ was used at 10⁻⁴ mol/L, all HaCaT cells were dead within a short period. Furthermore, recorded OD₄₅₀ values were significantly different compared with those for the negative control group (P<0.010). Our preliminary experimental results showed that HaCaT cell confluence increased gradually after inoculation and entered the plateau stage after 72 h in this assay system. Therefore, the difference between groups at 96 and 120 h could not be observed under these circumstances (P>0.05). Morphological analysis revealed that the cells in the negative control group and those treated with 10⁻⁸ or 10⁻⁷ mol/L Ag⁺ were polygonal-typical of keratinocytes (Fig. 1F) and exhibited roughly equivalent degrees of cell confluence. The cultures treated with 10⁻⁵ or 10⁻⁶ mol/L Ag⁺ contained a large proportion of polygonal cells and higher degrees of cell confluence than those in the negative control group (10⁻⁸ or 10⁻⁷ mol/L groups). For the cultures treated with 10⁻⁴ mol/L Ag⁺, very few cells were apparent, while those that remained were small. In addition, shrinkage of the imperfect cell membrane was observed.

Effects of Ag⁺ on intracellular ROS

The RFI of intracellular DCFH-DA indirectly reflects the production of ROS (Fig. 2). Compared with that in the negative control group, the ROS levels in the cells significantly increased with RFIs of 120.13%–192.97% after treatment with ROSup (P<0.01) and with RFIs of 106.11%–109.62% and 108.93%–115.28% after treatment with 10⁻⁸ and 10⁻⁷ mol/L Ag⁺, respectively. The RFI of DCFH-DA rapidly increased to 115.69%±10.20% and 120.69%±9.39% after 5 min in the cultures treated with 10⁻⁶ and 10⁻⁵ mol/L Ag⁺, respectively. The maximum RFIs were 129.05%±8.25% and 133.05%±6.95% at 60 min. The RFIs in the cultures treated with 10⁻⁴ mol/L Ag⁺ fluctuated between 105.53%±5.60% and 112.16%±6.10%, which were significantly lower than those in the positive controls and cultures treated with 10⁻⁵ or 10⁻⁶ mol/L Ag⁺ at all time points (P<0.05). A positive correlation (R= 0.900, P = 0.037) was found between the relative proliferation of HaCaT cells and increased intracellular ROS generation as the concentration of Ag⁺ increased when Ag⁺ concentration was below 10⁻⁴ mol/L.

We used MitoTracker^® Red CMXRos and confocal microscopy to visualize mitochondria in HaCaT cells (Fig. 2G). Fluorescence corresponding to the presence of DCFH-DA increased to a greater extent in the cultures treated with 10⁻⁶ and 10⁻⁵ mol/L Ag⁺ than in the negative controls. The RFI of DCFH-DA increased with increasing Ag⁺ concentration. Fluorescence was the strongest in the cultures treated with ROSup. Fluorescent signals were indicative of DCFH-DA localized to the same regions as mitochondria, confirming that ROS were mainly produced by mitochondria.

NAC affects the proliferation of HaCaT cells following treatment with Ag⁺

The treatment of HaCaT cells with 5 mmol/L NAC alone exerted no obvious effect on their growth (Fig. 3). However, the relative proliferation of cells treated with 10⁻⁶ or 10⁻⁵ mol/L Ag⁺ was 106.05%±2.14% (P = 0.005) or 116.32%±7.17% (P = 0.010), respectively, which was significantly different with that of the negative control group (100%). The relative proliferation of HaCaT cells following the addition of NAC was 97.65%±3.01% for the cultures initially treated with 10⁻⁶ mol/L Ag⁺ and 101.21%±5.64% for the cultures initially treated with 10⁻⁵ mol/L Ag⁺. These differences were significant in comparison with the cultures treated with 10⁻⁶ (P = 0.007) or 10⁻⁵ mol/L (P = 0.003) Ag⁺ alone but similar to those in the negative controls, which were not significant (P>0.05).

Effects of NAC on ROS production in HaCaT cells

Following treatment with 5 mmol/L NAC, the RFI of DCFH-DA at each time point was less than that at the corresponding time point of the negative control (Fig. 4A). The addition of 10⁻⁵ or 10⁻⁶ mol/L Ag⁺ increased the RFI at 5–60 min (P = 0.004) or 10–60 min (P = 0.004), respectively, and then peaked at 60 min. For cultures treated with 5 mmol/L NAC and 10⁻⁵ or 10⁻⁶ mol/L Ag⁺, the RFI of DCFH-DA was significantly decreased in comparison with cultures treated with Ag⁺ only. Our findings show that NAC exerted obvious antagonistic effects on Ag⁺ to stimulate ROS production in HaCaT cells. NAC exerted obvious antagonistic effects on Ag⁺ to stimulate ROS production in HaCaT cells. Confocal microscopy revealed that the RFI of DCFH-DA in HaCaT cells obviously decreased 60 min after Ag⁺ and NAC treatment compared with the cultures treated with Ag⁺ only, with no observed significant effects on mitochondria (Fig. 4G).

Effects of NAC and Ag⁺ on BrdU incorporation and PCNA expression in HaCaT cells

The proportion of BrdU-positive cells was determined 48 h after the treatment of HaCaT cells with NAC and/or Ag⁺. A greater number of BrdU-positive cells was observed in the cultures treated with 10⁻⁶ (52.94%±1.57%, P = 0.033) or 10⁻⁵ mol/L Ag⁺ (60.32%±4.20%, P = 0.015) than in the negative control cultures (45.48%±2.95%) (Fig. 5A). Following the addition of 5 mmol/L NAC, we observed a significant decrease in the percentage of BrdU-positive cells in the cultures exposed to 10⁻⁶ (P = 0.030) or 10⁻⁵ mol/L Ag⁺ (P = 0.010). The difference in the number of BrdU-positive cells between these cultures and the negative control cultures was not significant (P = 0.639 and 0.680).

We used immunocytochemical techniques to determine the presence of PCNA because its presence is an indirect indicator of cellular proliferation (Fig. 5B). Similar to our BrdU assay results, the fluorescence intensity of PCNA staining significantly increased in the cultures treated with 10⁻⁶ (P = 0.006) or 10⁻⁵ mol/L Ag⁺ (P = 0.008) than in the negative control cultures. The addition of 5 mmol/L NAC significantly decreased the PCNA fluorescence intensity in the cultures exposed to 10⁻⁶ (P = 0.006) or 10⁻⁵ mol/L Ag⁺ (P = 0.012) compared with the cultures that were not treated with NAC. No significant difference (P = 0.384 and 0.056) in PCNA intensity was observed between the NAC-treated cultures and negative control cultures. Confocal microscopy revealed the co-localization of BrdU and PCNA in the DAPI-stained nuclei (Fig. 5C). Compared with the negative controls, we observed an increase in the number of BrdU-positive cells and PCNA-positive cells following treatment with 10⁻⁶ or 10⁻⁵ mol/L Ag⁺ and a decrease in the number of BrdU-positive cells and PCNA-positive cells following coculture with NAC.

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

The proportion rates of HaCaT cells in the negative control cultures in the G₁ and S phases of the cell cycle were 48.23%±5.50% and 47.33%±4.50%, respectively, with the proliferation index (PI) of 51.77±5.49 (Table 1 and Fig. 6A). Treatment with 10⁻⁵ mol/L Ag⁺ (Fig. 6B) resulted in 39.91%±2.23% (P = 0.031) of cells in the G₁ phase and 53.46%±5.96% (P = 0.152) in the S phase, with 60.10±2.23 (P = 0.031) for PI. Treatment with 5 mmol/L NAC and 10⁻⁵ mol/L Ag⁺ increased the proportion of cells in the G₁ phase (50.15%±6.72%, P = 0.674) and decreased the percentage of cells in the S phase (46.84%±6.32%, P = 0.903), with PI decreasing to 49.85±6.71 (P = 0.674). These differences were significant when compared with the cultures treated with 10⁻⁵ mol/L Ag⁺ (P = 0.027) alone but were not significant compared with the negative controls (P = 0.674). Compared with those in the negative controls, all cell cycle indexes were not significantly altered in the cultures treated with 10⁻⁶mol/L Ag⁺ and/or 5 mmol/L NAC for 48 h.

Discussion

We reported for the first time that the sub-cytotoxic concentrations of Ag⁺ promote the proliferation of human keratinocytes by inducing ROS generation. Additionally, we confirmed that high concentrations of Ag⁺ are cytotoxic to HaCaT cells, as postulated by other scholars [¹¹,¹²,²¹,²³]. We also observed the effects of the active oxygen scavenger NAC on human keratinocytes exposed to Ag⁺. Our findings verify that the effect of Ag⁺ on promoting the proliferation of human keratinocytes is closely related to its role in inducing an appropriate production of ROS.

Burd et al. [⁷] and Collins et al. [¹²] reported that the concentrations of Ag⁺≥10⁻⁴ mol/L are cytotoxic to primary cultures of human keratinocytes. However, they failed to examine or report the effects of sub-cytotoxic Ag⁺ concentrations. Our results showed that the concentrations of Ag⁺≥10⁻⁴ mol/L were cytotoxic to HaCaT cells, which are similar to those of previous reports. However, the sub-cytotoxic concentrations of Ag⁺ (10⁻⁶ and 10⁻⁵ mol/L) promoted the proliferation of HaCaT cells between 24–72 h after addition to cultures. This finding was in contrast to the general belief that Ag⁺ is cytotoxic to cells. We subsequently sought to clarify the underlying mechanisms through which cellular proliferation is promoted.

Ag⁺ can alter the cellular redox state and induce ROS generation, likely leading to its cytotoxic effects [¹²,²¹]. Our results confirmed that Ag⁺ plays a significant role in promoting ROS generation within HaCaT cells. However, the levels of ROS generation did not increase in line with increasing concentrations of Ag⁺. The levels of ROS in HaCaT cells were higher in the cultures treated with 10⁻⁴ mol/L Ag⁺ than in the negative control cultures. However, ROS levels were significantly lower in the cultures treated with 10⁻⁴ Ag⁺ than in the cultures treated with 10⁻⁵ or 10⁻⁶ mol/L Ag⁺. This finding is possible because the combination of excess Ag⁺ with protein sulfhydryls can lead to protein deactivation and rapid destruction of the cell membrane structure. This condition would subsequently result in cell death and inability to produce ROS. Although Collins et al. [¹²] and Miyayama et al. [²¹] showed the influence of Ag⁺ on ROS generation in E. coli and human BEAS-2B cells, they did not reveal a dose–effect relationship between intracellular ROS levels and cell proliferation. The relative proliferation of HaCaT cells was enhanced markedly along with the increase in intracellular ROS generation when the cultures were treated with Ag⁺ at concentrations below 10⁻⁴ mol/L. This result could possibly provide a clue for explaining the ROS-induced proliferation of human keratinocytes following exposure to the sub-cytotoxic concentrations of Ag⁺.

ROS are considered a “double-edged sword” with respect to the regulation of physiological processes in the cell. High concentrations of ROS are involved in various pathological processes, including ischemia-reperfusion injury, cancer, atherosclerosis, and aging. Low concentrations of ROS play an important role in intracellular signal transduction and are necessary for cell proliferation and differentiation [²⁴–²⁶]. Treatment with sub-cytotoxic concentrations of Ag⁺ increased intracellular ROS levels by 9.62%–15.28% at 60 min but exerted no significant effects on cell proliferation. However, an increase of 29.05%–33.05% in ROS levels promoted cellular proliferation. The initial concentration range of ROS reflects “physiological concentration,” whereas a 29.05%–33.05% increase indicates the level of ROS required to promote proliferation. We used NAC to investigate the relationship between increased ROS levels and cell proliferation. NAC is a compound containing sulfur groups (–SH) that can easily pass across the cell membrane, deacetylate, and form L–cysteine, which is the precursor of the antioxidant glutathione. Glutathione is widely used clinically to treat paracetamol poisoning, ischemia–reperfusion cardiac injury, bronchitis, heavy metal toxicity, and psychiatric disorders, among others, as well as for a wide range of applications in research [²⁷,²⁸]. After treatment with 5 mmol/L NAC, the proliferative activities of HaCaT cells that had been promoted by Ag⁺ were reduced. Thus, NAC suppressed the effects of sub-cytotoxic Ag⁺ concentrations. Therefore, we hypothesized that sub-cytotoxic concentrations of Ag⁺ promote cellular proliferation by inducing moderate increases in ROS levels. The specific molecular mechanisms of action involved should be further investigated.

We used the CCK-8 method to determine cell proliferation [²⁹] in conjunction with BrdU incorporation assays and immunocytochemical detection of PCNA. BrdU incorporation allowed us to determine the rate of DNA synthesis and cell proliferation activity [³⁰]. Previous studies confirmed that low-energy laser irradiation, ionizing radiation, or direct exposure to exogenous O₂^– and H₂O₂ can moderately increase ROS generation and promote the proliferation of mouse embryonic fibroblasts [³¹], rat osteoblasts (MC-3T3E1), human smooth muscle cells, fibroblasts, prostate cancer cells [³²], and bovine artery endothelial cells [³³]. Our recent pre-experiments also showed similar effects when low concentrations of manganese and copper ions were added to the cell culture medium. Therefore, we believe that low concentrations of ROS from all sources exert a positive effect on cell proliferation, and silver ions promote cell proliferation through adequate ROS production rather than by silver ions themselves. ROS possibly exert effects on Ras, Rac, MEK, or MKK. Thus, ROS can activate ERK1/2 and JNK pathways downstream, upregulating the expression of transcription factors, including NF-kB, c-fos, and c-jun, and subsequently promoting proliferation [²⁶]. We propose that sub-cytotoxic Ag⁺ concentrations activate the related signaling pathways by upregulating intracellular ROS levels and enhancing intracellular DNA synthesis, manifesting as a higher proportion of BrdU-positive cells. Treatment with NAC reduces the number of BrdU-positive cells because it eliminates ROS and inhibits related signaling pathways. Therefore, the proliferating and promoting effects of Ag⁺ can be suppressed.

The expression of PCNA, an accessory protein of DNA polymerase, is regulated by the E2F transcription factor complex. PCNA expression during the G₀ phase and the start of the G₁ phase of the cell cycle is not normally observed. However, PCNA expression significantly increases in the late G₁ phase, peaks during the S phase, and then gradually declines during the G₂ and M phases. These quantitative changes are consistent with the levels of DNA synthesis, with the expression of PCNA in cells considered a sensitive index for proliferation [³⁴]. The upregulation of PCNA protein expression in the HaCaT cells treated with sub-cytotoxic concentrations of Ag⁺ confirms the effects of Ag⁺ on cellular proliferation. Increased PCNA expression indirectly affects cell cycle because it interacts with various cell cycle proteins, including cyclin, cyclin kinase, and cyclin kinase inhibitory proteins, during each phase of the cell cycle [³⁵], with a significant increase in the cell proliferation index. Although the number of S cells did not show statistical difference due to the different detection methods and time points among the treatment groups, the trend and upregulation of PCNA expression were completely consistent. NAC acts as an antagonist of proliferative effects conferred by Ag⁺ by inhibiting the expression of PCNA.

In conclusion, sub-cytotoxic concentrations of Ag⁺, which do not affect the intracellular redox equilibrium, induce appropriately increased levels of ROS. This phenomenon activates downstream signaling pathways, enhances mitochondrial metabolism, promotes DNA replication, and increases PCNA synthesis in human keratinocytes. Increased levels of ROS significantly decrease the number of cells in the G₁ phase but increase the number of cells in the S phase. The elimination of ROS using NAC suppresses proliferation induced by sub-cytotoxic concentrations of Ag⁺. Further investigations are required to fully elucidate the relevant signaling molecules and mechanisms involved in cellular proliferation promoted by Ag⁺.

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