Subjects
Fifteen healthy volunteers aged 19–35 years were enrolled in this trial. This study was approved by the institutional review board of the Yonsei University College of Medicine (IRB No. 3-2019-0239). All methods were carried out in accordance with the Declaration of Helsinki and Good Clinical Practice as defined under the Korea Food & Drug Administration and the International Conference on Harmonisation guidelines. The demographic and baseline characteristics are shown in Table 1. Written informed consent was obtained from all participants prior to inclusion in the study (ClinicalTrials.gov identifier: NCT04917835, 08/06/2021). Before enrollment, casual sebum levels at the surface of the forehead and on the right and left cheeks were measured using a Sebumeter® (SM815; Courage + Khazaka Electronic GmbH, Cologne, Germany). Skin type was classified based on sebum measurements according to the manufacturer’s guidelines as follows: > 220 µg/cm2 on forehead and > 180 µg/cm2 on cheeks, oily type; 100–220 µg/cm2 on forehead and 70–180 µg/cm2 on cheeks, normal type; < 100 µg/cm2 on forehead and < 70 µg/cm2 on cheeks, dry type. Only participants with oily skin type were enrolled in the study. At baseline, the mean (± standard deviation [SD]) casual sebum levels on the forehead and both cheeks were 238.2 ± 58.18 and 191.7 ± 51.66 mg/cm2, respectively. All participants had Fitzpatrick’s skin phototype III and mild acne vulgaris (grades 1–2 using the Korean acne grading system)20. Volunteers were excluded from the study if they had a history of surgical or cosmetic procedures or who had taken systemic retinoids within the prior 6 months; had been treated with topical retinoids within the prior 4 weeks; had acne of grade 3 or higher; had a concurrent systemic disease; had a history of hypertrophic scars or keloids; or were pregnant. To avoid diurnal variation, sebum levels were measured between and 10:00 and 12:00 h. Participants were instructed not to wear cosmetics for 2 h before the measurements. All measurements were made by the same trained physician in the same room under conditions of 20–22°C and 20–40% humidity conditions after 30 min acclimatization.
Generation of argon-and nitrogen-NTAPP pulses and clinical treatment
An NTAPP generator (PlaDuo™; Shenb Co., Ltd., Seoul, Korea) was utilized to generate argon- and nitrogen-NTAPP pulses from inert gaseous sources. Each unexcited gaseous source was loaded at the distal end of the handpiece, and then 2.45 GHz microwave energy converted the loaded sources to plasma in the nozzle. The treatment settings in terms of loading volume and pulse duration were determined according to energy settings; a loading volume of 0.12–0.67 mL/pulse and a pulse duration of 4–18 ms at an energy of 0.12–0.75 J/pulse for argon plasma, and 0.2–1.42 mL/pulse and 5–36 ms at 0.5–4 J/pulse for nitrogen gas.
As approved by the ethics committee, participants underwent three treatment sessions at 1-week intervals. Two passes of argon-NTAPP treatment and two passes of nitrogen-NTAPP treatment were sequentially performed in each session. The forehead, nose, and both cheek areas were treated with two passes of argon- and nitrogen-NTAPP pulses delivered at a pulse energy of 0.8 J/pulse with a 12 ms of pulse duration and at a pulse energy of 0.75 J/pulse delivered with a 7 ms of pulse duration, respectively, without pretreatment with topical anesthetic cream. The nozzle diameter was 5 mm, and the distance from the nozzle tip to the skin was 10 mm. The treated areas were cooled with icepacks immediately after treatment.
All participants were followed up 2, 4, and 8 weeks after the last session of NTAPP treatment. To investigate adverse events related to plasma treatment, patient symptoms were assessed by physicians. The extent of erythema, edema, pain, oozing, desquamation, postinflammatory hyperpigmentation, and scarring were scored at each visit by grading on a visual analog scale of 0 to 3 (0: absent; 1: mild; 2: moderate; 3: severe).
Measurement of porphyrin
After face washing and 30 min of acclimatization, UV photography was performed using a Mark-Vu® skin analysis imaging system (PSI Plus, Suwon, Korea) at baseline (week 0) and 2 weeks after the third session of plasma treatment (week 2). Sebum and porphyrins emit blue and orange fluorescence from UV light sources, respectively. First, to analyze the amount of sebum in the U zone (cheek area and forehead) and the partial T zone (nose), the ratio of the area occupied by blue fluorescence was calculated. The amount of porphyrin was calculated by splitting each RGB channel and then expressed as a percentage of the total sebum amount.
Measurement of facial sebum levels
Next, the casual sebum level was measured using a Sebumeter® in the forehead (mid-glabella) and the right and left cheeks (over the zygoma at the mid-pupillary line). The rate of sebum excretion, which represents the amount of facial sebum excretion for 60 min, was also investigated. Baseline sebum was measured immediately after washing. The subjects then remained in the same room under a controlled environment for 60 min, after which the second sebum level (at 60 min) was obtained. The sebum excretion rate was determined by calculating the difference between sebum levels at baseline and 60 min.
Skin biopsy, hematoxylin and eosin staining, immunohistochemistry, and immunofluorescence staining
Skin punch biopsies (3 mm) were obtained from the zygomatic area of two of 15 volunteers who completed the study, and 2 weeks after argon- and nitrogen-NTAPP treatment. Paraffin-embedded tissue sections (4 µm) were deparaffinized in xylene, rehydrated in an ethanol series, and incubated in blocking buffer (Novocastra, Newcastle upon Tyne, UK; 3% hydrogen peroxide) for 20 min at room temperature. Slides were incubated overnight at 4°C with a rabbit anti-mouse Ki67 antibody (1:200; Abcam, Cambridge, MA, USA; #ab15580), a rabbit polyclonal anti-4-Hydroxynonenal (4-HNE) antibody (1:100; Abcam, Cambridge, MA, USA; #ab46545), and a rabbit polyclonal anti-PPAR gamma antibody (1:100; Abcam, Cambridge, MA, USA; #ab59256), and then washed three times with phosphate buffered saline (PBS). The slides were incubated with HRP-conjugated anti-rabbit IgG (Dako, Carpinteria, CA, USA; #K4063) at room temperature for 60 min. To stain for Ki67, the slides were treated with diaminobenzidine for 5–10 min after washing and then washed again. Slides were stained with hematoxylin and eosin. Alexa Fluor 488-conjugated rabbit anti-mouse IgG (Thermo Fisher Scientific) was used as a secondary antibody for 4-HNE and PPARɣ staining. Images were captured using an LSM 780 confocal microscope (Carl Zeiss).
Sebocyte culture and NTAPP treatment
The immortalized human SZ95 sebaceous gland cell line16 was cultured in Dulbecco’s modified Eagle medium (DMEM)/F-12 culture medium supplemented with 2 mM GlutaMAX I, 10 µg/mL gentamicin, 50 ng/mL human epidermal growth factor, 10% fetal bovine serum (FBS), and 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (all purchased from Gibco BRL, Rockville, MD, USA) and maintained at 37°C and 5% CO2. The medium was replaced every 2 days. SZ95 sebocytes were seeded into 6-well plates and allowed to reach approximately 90% confluence overnight. To stimulate lipogenesis, sebocytes were treated with a combination of 2 × 10− 8 M testosterone and 10− 4 M linoleic acid (T/LA) for 48 h. All compounds were diluted in dimethyl sulfoxide (DMSO), and then diluted with culture medium (the final concentration of DMSO was 0.1%). Argon- or nitrogen-NTAPP pulses were delivered to sebocytes immediately or 24 h after treatment with T/LA. The 20 or 60 passes of argon-NTAPP pulse at an energy of 0.75 J and a pulse duration of 11 ms, or 5 or 10 passes of nitrogen-NTAPP pulse at an energy of 1.5 J and a pulse duration of 13 ms were administered to each well at a 5 mm distance. All controls were maintained in culture media at room temperature during the experiment to ensure equivalent treatment conditions.
Assessment of cell viability
SZ95 sebocytes were seeded into six-well plates at a density of 2 × 105 cells/well and then cultured overnight. Cells were irradiated with 20 or 60 pulses of argon-NTAPP at an energy of 0.75 J, and 5 or 10 pulses of nitrogen-NTAPP at an energy of 1.5 J and then incubated for 48 h. The cells were then incubated with 5 mg/mL of MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) for 2 h. The MTT solution was removed and then 200 µL of DMSO was added to dissolve the formazan crystals. The optical density (OD) was measured at 540 nm using a spectrophotometer.
Immunocytochemistry
SZ95 sebocytes were grown on coverslips, fixed in acetone, permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) in PBS, and then incubated with a rabbit anti-mouse Ki67 (1:200; Abcam; #AB15580) antibody for 60 min at 37°C (dilution 1:200). Slides were subsequently incubated with a goat anti-rabbit FITC-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) (dilution 1:100) for 30 min at room temperature, and nuclei were visualized with propidium iodide (PI) (Vector). The cells were examined using an LSM 780 confocal microscope (Carl Zeiss).
Determination of intracellular lipids
For the quantitative measurement of sebaceous lipids, SZ95 sebocytes (15,000 cells/well) were cultured in 96-well plates (Greiner Bio One, Frickenhausen, Germany) in quadruplicate and then treated with T/LA with or without plasma pulse treatment. Supernatants were then discarded, and 100 µL of 1 µg/mL Nile red (Sigma-Aldrich) solution was added to each well. The plates were incubated at 37°C for 20 min, and the emitted fluorescence was measured using a Molecular Devices FlexStation 384II fluorescence image microplate reader (FLIPR, Molecular Devices, San Francisco, CA, USA). The results are presented as percentages of absolute fluorescence units compared with the controls, at excitation and emission wavelengths of 485 and 565 nm, respectively, for neutral lipids. To detect lipid droplets by microscopy, 4% PFA-fixed cells were incubated with 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene 493/503 fluorescence lipid stain (10 µg/mL in PBS; BODIPY; Thermo Fisher Scientific, Waltham, MA, USA) for 7 min at 37°C. After incubation, the slides were washed with 0.5% BSA-PBS and imaged under fluorescence microscopy. Images were captured using a LSM 780 confocal microscope (Carl Zeiss).
Real-time quantitative polymerase chain reaction
RNA was extracted from SZ95 sebocytes using TRIzol reagent (Thermo Fisher Scientific), according to the manufacturer’s protocol. RNA was quantified using a NanoDrop 2000c (Thermo Fisher Scientific) and cDNA was synthesized using a cDNA Synthesis kit (Thermo Fisher Scientific). TaqMan real-time polymerase chain reaction (PCR) assays were performed to analyze mRNA levels (Applied Biosystems, Foster City, CA, USA). TaqMan probes for PPARγ (Hs01115514_m1, Thermo Fisher Scientific) and sterol regulatory element binding protein-1 (SREBP1) (Hs01088678_g1, Thermo Fisher Scientific) were used. The relative expression of each gene was normalized to the mean level of GAPDH mRNA (Hs02786624_g1, Thermo Fisher Scientific).
Statistical analysis
Statistical analyses were performed using Prism 6 (GraphPad) and R version 3.4.3. All tests were two-tailed. An unpaired t-test was used to compare data between the two groups. Statistical significance was set at *P < 0.05, **P < 0.01, with error bars used to indicate the standard error of the mean (SEM).