Cytoprotective effects of mild plasma-activated medium against oxidative stress in human skin fibroblasts

Non-thermal atmospheric pressure plasma (NTAPP) has recently been applied to living cells and tissues and has emerged as a novel technology for medical applications. NTAPP affects cells not only directly, but also indirectly with previously prepared plasma-activated medium (PAM). The objective of this study was to demonstrate the preconditioning effects of “mild PAM” which was prepared under relatively mild conditions, on fibroblasts against cellular injury generated by a high dose of hydrogen peroxide (H2O2). We observed the preconditioning effects of mild PAM containing approximately 50 μM H2O2. Hydrogen peroxide needs to be the main active species in mild PAM for it to exert preconditioning effects because the addition of catalase to mild PAM eliminated these effects. The nuclear translocation and recruitment of nuclear factor erythroid 2-related factor 2 (Nrf2) to antioxidant response elements (ARE) in heme oxygenase 1 (HO-1) promoters and the up-regulation of HO-1 were detected in fibroblasts treated with mild PAM. The addition of ZnPP, a HO-1-specific inhibitor, or the knockdown of Nrf2 completely abrogated the preconditioning effects. Our results demonstrate that mild PAM protects fibroblasts from oxidative stress by up-regulating HO-1, and the H2O2-induced activation of the Nrf2-ARE pathway needs to be involved in this reaction.

NTAPP has been reported to affect cells not only directly, but also indirectly with previously prepared plasma-activated medium (PAM) 6,[21][22][23] . The relatively short-lived ROS and RNS produced in medium by NTAPP irradiation are converted to other relatively long-lived species such as H 2 O 2 , nitrite/nitrate (NOx), and other unknown species 21,24,25 . PAM has recently been shown to induce apoptosis in cancer cells 6,[25][26][27][28][29][30] . On the other hand, the effects of PAM prepared under relatively mild conditions, termed mild PAM, on normal cells are currently unclear. The stimulation of Phase II antioxidant pathways in keratinocytes by PAM has recently been reported 31 .
The objective of this study was to demonstrate the preconditioning effects of mild PAM on fibroblasts against cellular injury generated by a high dose of H 2 O 2 as an oxidative stress inducer and elucidate the involvement of the Keap1-Nrf2-ARE signaling pathway in this cytoprotection.

Reductions in H 2 O 2 -induced cell injury by the pretreatment with mild PAM. NTAPP irradiation
produced H 2 O 2 in DMEM in irradiation time-dependent manners under our experimental conditions, as shown in Fig. 1a. In the present study, fibroblasts were treated with mild PAM for 6 h and then incubated with culture medium for 18 h. Fibroblasts were then treated with 500 μ M H 2 O 2 as strong oxidative stress for 4 h, as shown in Fig. 1b left panel, except in specifically described experiments. Cell injury induced by the treatment with 500 μ M fibroblasts were pretreated with mild PAM for 6 h in a CO 2 incubator. After the exchange of mild PAM to DMEM-10% FCS, fibroblasts were cultured for 18 h in a CO 2 incubator. Fibroblasts were then treated with 500 μ M H 2 O 2 in DMEM for 4 h in a CO 2 incubator, followed by an assay for LDH released into conditioned medium. (b, right) Fibroblasts were pretreated with mild PAM prepared by the irradiation of NTAPP for the indicated times. Control (Ct) fibroblasts were incubated with DMEM for 4 h instead of 500 μ M H 2 O 2 in DMEM. (c) After the pretreatment with mild PAM or DMEM (vehicle), and followed by the incubation with DMEM-10% FCS, fibroblasts were treated with DMEM with or without 250, 500, and 1000 μ M H 2 O 2 for 4 h, followed by the LDH assay. (d) Fibroblasts were pretreated with mild PAM, 50 μ M H 2 O 2 in DMEM, or DMEM (vehicle) for 6 h, followed by the procedure described above. (e) Fibroblasts were treated with mild PAM in the presence or absence of catalase (50 U/mL) or DMEM (vehicle) for 6 h, followed by the procedure described above. (f) Fibroblasts were treated with mild PAM, 100 μ M H 2 O 2 in DMEM, 500 μ M H 2 O 2 in DMEM, or DMEM (vehicle) for the indicated times, followed by the procedure described above. (g, left) Concentrations of nitrite in mild PAM prepared by irradiation with NTAPP for the indicated times. (g, right) Fibroblasts were pretreated with mild PAM, 900 μ M nitrite in DMEM, or DMEM (vehicle) for 6 h. (h, left) A549 cells were pretreated with mild PAM or DMEM (vehicle) for 6 h, followed by the procedure described above. (h, right) A549 cells were treated with mild PAM or DMEM (vehicle) for 6 h, and HO-1 mRNA levels were then measured. Data are shown as the mean ± SD (n = 3), *p < 0.05, **p < 0.01 vs. the control, # p < 0.05, ## p < 0.01 vs. vehicle, $ p < 0.05, NS not significant. H 2 O 2 was significantly suppressed by the pretreatment with mild PAM prepared by plasma irradiation for 2 min, whereas mild PAM prepared by irradiating for 1 min did not affect cell injury, as shown in Fig. 1b right panel. On the other hand, (mild) PAM prepared by irradiating for 3 min induced cell injury, even without the H 2 O 2 treatment. Fibroblasts pretreated with mild PAM significantly resisted cytotoxicity induced by H 2 O 2 at concentrations of 500 or 1000 μ M (Fig. 1c). H 2 O 2 is known to be the main active species in PAM [24][25][26][27] , and mild PAM prepared by an irradiation time for 2 min contained approximately 50 μ M H 2 O 2 , as shown in Fig. 1a. The pretreatment with 50 μ M H 2 O 2 -supplemented DMEM provided a similar degree of suppression against H 2 O 2 -induced cellular injury to mild PAM (Fig. 1d). Moreover, fibroblasts pretreated with mild PAM supplemented with catalase (50 U/ml) did not resist cell injury (Fig. 1e). The preconditioning effects of mild PAM depend on its H 2 O 2 concentration and pretreatment time. Cell injury was significantly and maximally suppressed by the pretreatment with mild PAM for 6 h and followed by 3 h, as shown in Fig. 1f left panel. On the other hand, 100 μ M H 2 O 2 -supplemented DMEM, the H 2 O 2 concentration of which was the same as that of (mild) PAM prepared by NTAPP irradiation for 3 min, exerted preconditioning effects by the pretreatment for 3 h. However, cell injury was induced by the pretreatment for 6 h. The pretreatment with 500 μ M H 2 O 2 -supplemented DMEM, the H 2 O 2 concentration of which was similar to that of PAM used in previous studies 25,27,28 , for 30 min suppressed the release of LDH (Fig. 1f right). However, cells detached from the culture dish following the pretreatment with 500 μ M H 2 O 2 -supplemented DMEM for 3 h.
Another main species in mild PAM is NOx, and NTAPP irradiation for 2 min produced approximately 900 μ M nitrite as NOx (Fig. 1g left). However, the pretreatment with 900 μ M nitrite-supplemented DMEM did not reduce cell injury (Fig. 1g right).
We previously reported that a PAM treatment effectively induced apoptosis in A549 human lung adenocarcinoma epithelial cells 25,27,28 . When A549 cells were treated with mild PAM according to the protocol shown in Fig. 1b left, mild PAM led to slightly greater cell injury than that with the vehicle, as shown in Fig. 1h left panel.

Induction of HO-1 by the treatment with mild PAM.
In order to elucidate the effects of mild PAM on the expression of HO-1, fibroblasts were treated with mild PAM for 6 h. Mild PAM prepared by NTAPP irradiation for 1 or 2 min increased the expression of HO-1 mRNA (Fig. 2a). On the other hand, (mild) PAM prepared by irradiation for 3 min decreased HO-1 mRNA levels. The mild PAM treatment for 6 h maximally enhanced the expression of HO-1 mRNA (Fig. 2b). The protein level of HO-1 in mild PAM-treated cells also increased ( Fig. 2c). HO-1 mRNA levels were significantly increased by the treatment with 50 μ M H 2 O 2 -supplemented DMEM, but were only moderately increased by that with 10 μ M H 2 O 2 -supplemented DMEM. HO-1 mRNA levels were decreased by the treatment with 100 μ M H 2 O 2 -supplemented DMEM (Fig. 2d). Mild PAM supplemented with catalase did not induce the expression of HO-1 mRNA, while 50 μ M H 2 O 2 -supplemented DMEM achieved the similar up-regulation of HO-1 mRNA to mild PAM. tert-butylhydroquinone (tBHQ), a known HO-1 enhancer, up-regulated HO-1 mRNA (Fig. 2e, left). On the other hand, the mRNA of HO-2, a constitutive enzyme, was not induced by the treatment with mild PAM (Fig. 2e right). These results suggest that the ability of mild PAM to induce HO-1 depends on H 2 O 2 . In an attempt to clarify whether HO-1 induced by the mild PAM treatment confers cytoprotection against toxicity induced by a high dose of H 2 O 2 , fibroblasts were cultured in the presence of the HO-1 inhibitor ZnPP (10 μ M) after a pretreatment with mild PAM. ZnPP eliminated the protective effects of mild PAM against H 2 O 2 -induced cell injury (Fig. 2f).
The treatment of A549 cells with mild PAM did not affect HO-1 mRNA levels ( Fig. 1h right panel).

Enhancement of the ARE-binding activity of Nrf2 in mild PAM-treated fibroblasts.
Nrf2 is a major transcription factor that binds to ARE in the promoter region of some antioxidative enzymes. When fibroblasts were treated with mild PAM for 6 h, the nuclear translocation of Nrf2 was detected (Fig. 3a). This Nrf2 nuclear translocation was also observed by fluorescence imaging with a confocal microscopic analysis (Fig. 3b).
In addition, ChIP assays showed an increase in Nrf2 binding to ARE in the HO-1 promoter (Fig. 3c). In order to elucidate whether Nrf2-mediated transcriptional activation is involved in the induction of HO-1 in response to mild PAM, we transfected Nrf2-specific siRNA (siNrf2) or control siRNA into fibroblasts and treated these cells with mild PAM for 6 h. We confirmed that the transfection of siNrf2 caused a marked reduction in Nrf2 in fibroblasts (Fig. 3d). siNrf2 attenuated the induction of HO-1 mRNA by the treatment with mild PAM (Fig. 3e). Moreover, mild PAM-induced cytoprotection against toxicity induced by a high dose of H 2 O 2 was significantly reduced by the transfection of siNrf2 (Fig. 3f).
We previously reported that a PAM treatment effectively induced apoptosis in A549 lung adenocarcinoma cells 25 Nitrite, another main species in mild PAM, did not exert preconditioning effects (Fig. 1g). Nitrite by itself has been suggested to not play a role in the antitumor effects of PAM 25,36 . However, nitrite may affect the potency of PAM and mild PAM because the cancer cell-killing ability of H 2 O 2 is reported to be synergistically enhanced by nitrite 36 .
Nrf2-mediated HO-1 induction was previously shown to play crucial roles in the anti-oxidative defense system [34][35][36][37][38][39] . HO catalyzes the degradation of heme to carbon monoxide, a vasoactive gas; biliverdin, an antioxidant; and free iron 40 . The HO system possesses antioxidative and anti-apoptotic properties, and may influence cell proliferation, differentiation, and migration 41 . Therefore, we postulated that the Nrf2-HO system may be involved in mild PAM-induced preconditioning effects. Among the three HO isoforms (HO-1, HO-2, and HO-3), HO-1 is the only inducible enzyme that plays a critical role in cytoprotection against oxidative stress, inflammation, and other noxious stimuli 37 . In the present study, mild PAM elevated HO-1 mRNA and protein levels, but not those of HO-2 mRNA (Fig. 2e). Moreover, 50 μ M H 2 O 2 significantly increased HO-1 mRNA levels (Fig. 2d). The induction of HO-1 mRNA was not observed when fibroblasts were treated with catalase-supplemented mild PAM (Fig. 2e,  left). These results suggest that H 2 O 2 in mild PAM is critical for the mild PAM-induced expression of HO-1. However, we cannot rule out the possibility that a mechanism other than the induction of HO-1 is involved in the preconditioning effects of the mild PAM pretreatment, and other unknown species in mild PAM may play a role in the induction of HO-1 because mild PAM prepared using NTAPP irradiation for 1 min increased HO-1 mRNA levels, but did not affect cell injury (Fig. 1b), and the induction of HO-1 by 10 μ M H 2 O 2 was moderate (Fig. 2d). The roles of HO-2 and HO-3 in cells currently remain unclear. HO-2 mRNA levels are low in human dermal fibroblasts, but high in keratinocytes, and HO-2 in keratinocytes plays a role in maintaining iron release from ferritin 42 . The result that mild PAM did not exert preconditioning effects in A549 cells was attributed to the absence of an increase in HO-1 mRNA levels (Fig. 1h); however, the underlying mechanisms remain unclear. HO-1 is positively regulated by the Keap1-Nrf2-ARE pathway 39,40,43 . In this study, we revealed the nuclear translocation ( Fig. 3a and b) and recruitment of Nrf2 to HO-1 promoters (Fig. 3c) in fibroblasts exposed to mild PAM for 6 h. The knockdown of Nrf2 attenuated the induction of HO-1 by mild PAM (Fig. 3e), suggesting that mild PAM up-regulates the Nrf2-dependent expression of HO-1. The up-regulation of HO-1 correlated with mild PAM-induced preconditioning effects against a high dose of H 2 O 2 (Figs 1 and 2). Our results showed that the addition of ZnPP, a HO-1 specific inhibitor (Fig. 2f) or knockdown of Nrf2 (Fig. 3f) completely abrogated the preconditioning effects of mild PAM. H 2 O 2 -induced Keap1-Nrf2-ARE signaling pathway is well known [18][19][20] . In this pathway, H 2 O 2 oxidize cysteine residues in Keap1 and change its conformation 18,44 . This conformational change weakens the interaction between Keap1 and Nrf2, and inhibits the subsequent ubiquitination and degradation of Nrf2. Therefore, Nrf2 is stabilized and translocated to the nucleus, and then binds to ARE in the HO-1 promoter. The phosphatidylinositol 3 kinase (PI3K)-Akt signaling pathway is known to activate Nrf2 33,45,46 and its downstream signaling induces HO-1 45 . H 2 O 2 has been identified as an activator of the PI3K-Akt signaling pathway 46 . The ARE binding activity of Nrf2 was measured using the ChIP assay. Fibroblasts were treated with mild PAM, tBHQ (20 μ M), or DMEM (vehicle) for 6 h. Their chromatins were cross-linked and immunoprecipitated with IgG or an anti-Nrf2 antibody (Ab), and DNAs were amplified using an ARE-specific primer. Data are shown as the mean ± SD (n = 3), *p < 0.05 vs. vehicle. (d) After siNrf2 and the siNC were transfected into fibroblasts for 24 h in a CO 2 incubator, whole cell extracts were prepared and followed by Western blotting for Nrf2. (e) After transfection of siNrf2 or siNC, cells were treated with mild PAM or DMEM (vehicle) for 6 h and HO-1 mRNA levels were then measured. Data are shown as the mean ± SD (n = 3), *p < 0.05, **p < 0.01 vs. vehicle, # p < 0.05. (f) After transfection of siNrf2 or siNC, cells were treated with mild PAM or DMEM (vehicle) for 6 h, followed by cultivation for 18 h, a treatment with or without 500 μ M H 2 O 2 for 4 h, and the LDH assay. Control (Ct) fibroblasts were incubated with DMEM for 4 h instead of 500 μ M H 2 O 2 in DMEM. Data are shown as the mean ± SD (n = 3), **p < 0.01 vs. vehicle, # p < 0.05.
ROS/RNS or their derived species are generally considered to be the main bioactive species in PAM. We speculated the involvement of other H 2 O 2 -cooperating species in PAM-induced apoptosis in cancer cells, whereas H 2 O 2 needs to be the main active species in the reaction 25 . Moreover, ROS-derived amino acid peroxides (organic peroxides) 1 and/or nitrites 36 may contribute to the potency of PAM. It is not currently possible to rule out the involvement of these species and other unknown species and latent mechanisms in the preconditioning effects of mild PAM.
A relatively low dose of plasma has been reported to induce the proliferation of cells 7,8 , and has potential for clinical applications, for example, wound healing 2,47,48 . On the other hand, the induction of HO-1 has also been reported to play a crucial role in wound healing 41,49,50 . Our results clearly demonstrate that the pretreatment with mild PAM protected fibroblasts from oxidative stress by up-regulating HO-1; tens of μ M of H 2 O 2 in mild PAM contribute to preconditioning effects; the H 2 O 2 -induced activation of the Nrf2-ARE signaling pathway needs to be involved in the up-regulation of HO-1 (Fig. 4). Although further investigations are needed, the results of the present study provide evidence for the anti-oxidative stress functions of mild PAM and its potential for clinical applications.
Preparation of mild PAM. The experimental set-up of the NTAPP irradiation system used in this study consisted of a power controller/gas flow regulator, argon gas cylinder, and NTAPP source head (PN-120 TPG, NU Global, Nagoya, Japan) and was the same as the system described previously [25][26][27][28] . The flow rate of argon gas was set at 2 standard liters/min. Mild PAM was prepared by exposing NTAPP to 4 ml of DMEM (Sigma 5796), without FCS and antibiotics, in 60-mm culture dishes (Nunc 150288). The distance between the plasma source and surface of the medium was fixed at L = 4 mm. The duration time for PAM irradiation was 2 min, except in the time-course experiment.
Assays to measure H 2 O 2 and nitrite concentrations. H 2 O 2 concentrations in mild PAM were assayed by a colorimetric method using 3-methyl-2-benzothiazolinone hydrazine hydrochloride, N,N-dimethylaniline, and horseradish peroxidase 25 . Nitrite as NOx was assayed by the Griss method 51 . The concentrations of H 2 O 2 and nitrite in mild PAM were measured immediately after its preparation and it was promptly used in experiments.
Cytotoxicity assay. The lactate dehydrogenase (LDH) assay was used to estimate cytotoxicity. Fibroblasts were seeded at 1.5 × 10 4 cells/well in a 96-well microplate (Nunc 167008), cultured for 24 h in a CO 2 incubator, and then used in experiments. Cells were treated with 80 μ l of mild PAM for 6 h in a CO 2 incubator. After the removal of mild PAM, cells were provided DMEM-10% FCS (80 μ l) and incubated for 18 h in a CO 2 incubator. After the removal of medium, cells were treated with 500 μ M H 2 O 2 in DMEM (80 μ l) as an oxidative stress for 4 h in a CO 2 incubator, and this was followed by an assay for LDH released into conditioned medium using a LDH cytotoxic test (Wako Pure Chemical, Osaka, Japan) according to the manufacturer's directions. solution was subsequently incubated with 20 μ l of Dynabeads Protein G (Invitrogen) at 4 °C for 2 h with shaking. After being incubated, the beads were sequentially washed with RIPA buffer I, RIPA buffer II (50 mM Tris-HCl, pH 8.0, containing 500 mM NaCl, 1 mM EDTA, 0.1% SDS, 0.1% deoxycholic acid, and proteinase inhibitors), and TE buffer (10 mM Tris-HCl, pH 8.0, containing 1 mM EDTA). The beads were suspended in 100 μ l of ChIP elution buffer (10 mM Tris-HCl, pH 8.0, containing 300 mM NaCl, 5 mM EDTA, and 0.5% SDS) with 1 μ l RNase (Roche Diagnostics, Mannheim, Germany) and incubated at 37 °C for 30 min, followed by the addition of 1 μ l proteinase K (Roche Diagnostics) and an incubation at 65 °C for 2 h. After phenol-chloroform extraction and ethanol precipitation, genomic DNA was dissolved in 20 μ l of TE buffer. The abundance of ARE in ChIP precipitates was quantified using a RT-PCR analysis. The primer sequences for ARE were sense 5′ -CCC TGC TGA GTA ATC CTT TCC CGA-3′ , antisense 5′ -ATG TCC CGA CTC CAG ACT CCA-3′ .
Data analysis. Data are presented as the mean ±SD of three experiments. Data were analyzed by Welch's t-test. A p value of less than 0.05 was considered significant.