PM2.5 was collected by TH-150C Medium flow air total suspended particulate sampler (Wuhan TianHong Environmental Protection Industry Co., Ltd., Wuhan, China) (100 1/min) in Wuhan urban air during July 2015. The sample was set on the rooftop of a building of the Institute of Atmospheric Research, School of Environmental Studies, China University of Geosciences (Wuhan, China), ~6 m above the ground. The filter was changed every 24 h using the air total particle sampler. Subsequent to sampling, the PM2.5 filter was placed under an ultraviolet lamp for 30 min and cut into two equal size pieces. The W-PM2.5 and NW-PM2.5 were isolated and prepared as previously described (23). The samples were collected and stored at −80°C until later use.

Immortalized human SZ95 sebaceous gland cells, (patented cell, gifted by Professor Christos C. Zouboulis, Department of Dermatology, Venereology, Allergology and Immunology, Dessau Medical Center, Theodore Fontane Medical University of Brandenburg, Germany) at passages 30–40 were cultured at 37°C in Sebomed^® basal medium (Biochrom GmbH, Berlin, Germany) supplemented with 10% fetal calf serum, Gibco 5 ng/ml human epidermal growth factor (Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 U/ml penicillin and 100 g/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc.). Various concentrations (1, 10, 100 and 250 µg/ml) of PM2.5 were prepared. For the exposure, cells were exposed to a W-PM2.5 or NW-PM2.5 for 48 h.

Following exposure for 48 h, cell viability was determined using CCK-8 assay kits (C0038; Shanghai Beyotime Biotechnology Co., Ltd., Shanghai, China) according to the manufacturer's instructions. CCK-8 solution (10 µl) was added to each well, followed by an incubation for 1–4 h. Absorbance was measured using a microplate reader at a wavelength of 450 nm. The cells treated with the solvent served as a control group and wells without cells served as the blank group. Cell viability was calculated according to the following formula: cell proliferation (%) = optical density (OD) of the experimental group/OD of the control group ×100.

SZ95 sebocytes were seeded in 96-well plates. Subsequent to exposure for 48 h, lactate dehydrogenase (LDH) activity was detected using a Pierce LDH cytotoxicity assay kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol.

SZ95 sebocytes were seeded into 6-well plates. Following exposure for 48 h with different concentrations of PM2.5 suspension, cells were collected using 0.25% trypsin and phosphate-buffered saline (PBS). Also, culture was centrifuged at 300 × g for 5 min at room temperature twice. After that precooled 90% ethanol was added. Then cells were resuspended at 4°C for 20 min and centrifuged at 300 × g for 5 min at room temperature. Cells were incubated with propidium iodide (PI) buffer [50 mg/ml containing ribonuclease A (50 ng/ml)] at room temperature and stained in the dark for 20 min at room temperature. Cell cycle distribution was analyzed using a BD Biosciences Flow Cytometer (BD Biosciences, San Jose, CA, USA).

SZ95 sebocytes were seeded in 24-well plates (5×10⁴ cells/well). After a 48-h exposure with different concentrations of PM2.5 suspension, cells were washed with PBS and fixed in 10% neutral formaldehyde for 30 min. The cells were then washed twice in distilled water for 2 min, stained with Oil Red O dye (AS1083; Wuhan Aspen Biotechnology Co., Ltd., Wuhan, China) (0.5% Oil Red dissolved in isopropanol, then diluted with distilled water at a ratio of 6:4) at room temperature in the dark for 10 min and washed in distilled water for 1 min. Subsequently, cells were counterstained with hematoxylin, and then sealed by glycerol gelatin for long-term preservation, and observed under an inverted Olympus microscope.

SZ95 sebocytes were seeded in 24-well plates (1×10⁵ cells/well). After 48-h exposure to different concentrations of PM2.5 suspension, the supernatant was collected at 4°C, centrifuged for 20 min at 1,000 × g at 4°C, and the supernatant was collected and stored at −20°C for subsequent assays. Concentrations of IL-1α, IL-6 and IL-8 were determined using commercial ELISA kits (E-EL-H0088c, E-EL-H0102c and E-EL-H0048c; ElabScience Biotechnology Co., Ltd., Wuhan, China) according to the manufacturer's instructions.

Immunofluorescent cytochemical analysis was performed using anti-AhR (17840-1-AP), anti-ARNT (14105-1-AP), anti-CYP1A1 (13241-1-AP) (all from Proteintech Group, Inc., Wuhan, China) and Cy3-conjugated anti-rabbit IgG antibodies (AS-1109; Wuhan Aspen Biotechnology Co., Ltd.). SZ95 sebocytes were seeded into 6-well plates. Following exposure for 48 h to various concentrations of PM2.5 suspension, coverslips were added to the plates and incubated with 5% CO₂ at 37°C until cells reached 80% confluence. Media was aspirated from the plates and washed 3 times with PBS. Cells were fixed with 4% paraformaldehyde for 30 min at room temperature. We then added Triton X-100 for 10 min and hydrogen peroxide solution at room temperature in the dark for 20 min; after each step, the cells were washed 3 times with PBS for 5 min. The cell culture was incubated with primary antibody overnight at 4°C and washed 3 times for 5 min with PBS. Stained with conjugated secondary antibody for 30 min at room temperature for 50 min and washed 3 times for 5 min with PBS. Nuclear staining was then performed with DAPI. Images of each slide were obtained using an optical microscope system (Axiomager; Carl Zeiss AG, Oberkochen, Germany). The protein expression levels of AhR, ARNT and CYP1A1 were quantified with an image analysis program (Q-imaging Pro-7; Olympus, Tokyo, Japan) from the microscope system.

Following exposure for 48 h to various concentrations of PM2.5 suspension, the adherent cells were washed with TBS buffer for 3 times. After drying the residual liquid, protease inhibitor was added for 3 min at room temperature. The cell total protein extraction reagent was then added for 5 min at room temperature. Cells were collected to 1.5 ml centrifuge tubes and centrifuged at 10,000 × g for 5 min at 4°C. The supernatant which was the total protein solution was collected at 4°C. The concentration of the proteins was determined using a BCA protein assay kit (AS1086; Wuhan Aspen Biotechnology Co., Ltd.). Total protein samples (ensure that the total protein amount of each sample was 40 µg) were separated with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), then transferred onto polyvinylidene fluoride membrane (EMD Millipore, Bedford, MA, USA), blocked with skimmed milk and incubated overnight at 4°C with primary antibodies against anti-AhR (dilution, 1:1,000), anti-ARNT (dilution, 1:500), CYP1A1 (dilution, 1:500) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (dilution, 1:1,000). The membranes were incubated with the secondary antibody (horseradish peroxidase goat anti rabbit; dilution, 1:10,000) for 30 min at room temperature, and the blots were detected using an enhanced chemiluminescence kit (Cell Signaling Technology, Inc., Danvers, MA, USA). GAPDH served as an internal control.

Results are expressed as the mean ± standard error of the mean. Statistical analysis was performed using two-way ANOVA and Dunnett's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

To evaluate the effects of PM2.5 exposure on the viability of SZ95 sebocytes, a CCK-8 assay was performed in the present study and the results are presented in Fig. 1. PM2.5 (NW-PM2.5 and W-PM2.5) exposure for 48 h significantly induced cell viability when compared with the control cells (P<0.01) (Fig. 1A and B). No significant difference was identified between the viability of NW-PM2.5- and W-PM2.5-exposed SZ95 sebocytes (P>0.05). In addition, the toxicity of NW-PM2.5 and W-PM2.5 exposure on SZ95 sebocytes was evaluated using a Pierce LDH cytotoxicity assay kit according to the manufacturer's instructions. Compared with the control cells, NW-PM2.5 exposure (10 µg/ml, P<0.05; 100 and 250 µg/ml, P<0.01) significantly increased LDH activity in a dose-dependent manner (Fig. 1C). W-PM2.5 exhibited similar effects on SZ95 sebocytes (Fig. 1D).

The effects of PM2.5 on the cell cycle were investigated (Fig. 2). Flow cytometry demonstrated that compared with the unexposed control cells, the number of cells in the G₁ phase were markedly increased in cells exposed to NW-PM2.5 (Fig. 2A). With increasing NW-PM2.5 concentrations, the cell rates in the G₁ phase also significantly increased. As illustrated in Fig. 2B, compared with the control cells, W-PM2.5 exposure markedly increased the number of cells in the G₁ phase in a dose-dependent manner.

As lipids secreted by sebaceous gland cells are important in the process of skin physiology involving cell structure, cohesion and desquamation as well as formation and function of a permeability barrier, the effects of PM2.5 exposure on lipid synthesis were examined in SZ95 sebocytes. Oil Red O staining indicated that NW-PM2.5 (Fig. 3A) and W-PM2.5 (Fig. 3B) at low concentrations promoted lipid synthesis, while high concentrations of NW-PM2.5 and W-PM2.5 markedly inhibited lipid synthesis in SZ95 sebocytes.

Whether PM2.5 exposure regulated pro-inflammatory cytokine secretion was investigated by ELISA of pro-inflammatory cytokines (IL-1α, IL-6 and IL-8). NW-PM2.5 (Fig. 4A, C and E) and W-PM2.5 (Fig. 4B, D and F) exposure dose-dependently elevated the levels of all the evaluated pro-inflammatory cytokines that were released by SZ95 sebocytes, when compared with the unexposed control cells.

In addition, whether AhR/CYP1A1 signaling is involved in the effect of PM2.5 exposure on SZ95 sebocytes was evaluated. Western blot analysis revealed that compared with unexposed control cells, SZ95 sebocyte exposure to NW-PM2.5 dose-dependently induced the elevation of protein expression levels of AhR, ARNT and CYP1A1. Similarly, the AhR, ARNT and CYP1A1 expression levels were reduced in the unexposed control cells, whereas the AhR, ARNT and CYP1A1 expression levels in SZ95 sebocytes were markedly increased in a dose-dependent manner by W-PM2.5 (Fig. 5).

Furthermore, immunocytochemical analysis (Fig. 6) demonstrated the localization of AhR, ARNT and CYP1A1 in PM2.5-exposed SZ95 sebocytes. Compared with unexposed control cells, significant AhR induction was observed in NW-PM2.5- (Fig. 6A) and W-PM2.5-exposed (Fig. 6B) cells. Similar to AhR, the staining for ARNT and CYP1A1 was significantly and dose-dependently increased in NW-PM2.5-exposed [ARNT (Fig. 6C); CYP1A1 (Fig. 6E)] and W-PM2.5-exposed [ARNT (Fig. 6D); CYP1A1 (Fig. 6F)] SZ95 sebocytes.