Here, 10 patients (6 females and 4 males, 36.3±4.1 years) who were non-smokers and had either not been treated with glucocorticoids (systemic or topical), antihistamines, non-steroidal anti-inflammatory drugs, or macrolide antibiotics for at least 1 month or who had ceased using the drug at least 1 month due to lack of alleviation or even aggravation of symptoms, were recruited from the Department of Otorhinolaryngology at Sun Yat-sen Memorial Hospital. All participants provided written informed consent in advance, and nasal polyp tissues were obtained during surgery. The study was approved by the Ethics Committee of Sun Yat-sen Memorial Hospital.

NPDFs were isolated from nasal polyp tissue from patients who had chronic rhinosinusitis with nasal polyps using a previously described method (14). Briefly, nasal polyp tissue was cut into pieces, re-suspended in DMEM/F12 containing 1 mg/ml collagenase I in a centrifuge tube, and then placed in a 5% CO₂ humidified atmosphere at 37°C. After 12 h, the tissues were re-suspended in DMEM/F12 with 10% FBS and cultured in a 5% CO₂-humidified atmosphere at 37°C, and the medium was changed every 48 h. Trypsin enzymatic digestion and differential attachment were used to isolate fibroblast cells.

The isolated fibroblasts were identified immunocytochemistry using vimentin as a positive marker and CK (pan) as a negative marker. After 3 rounds of washing with PBS, the cells were incubated for 30 min with 5% BSA to block non-specific sites. The coverslips were then incubated with primary antibody (vimentin or CK) (1:200 dilution) in 1% BSA in a humidified chamber overnight at 4°C. Then the coverslips were incubated with HRP conjugated anti-rabbit secondary antibody for 30 min, and washed 3 times for 5 min each with PBS. DAB substrate was used to detect HRP activity. Another group of NPDFs were grown in 6-well plates using DMEM/F12 containing 10% FBS with or without 100, 200, or 400 µM bleomycin A5 for 48 h in a 5% CO₂ humidified atmosphere at 37°C to assess the influence of bleomycin A5 on NPDFs.

Cultured NPDFs (1×10⁴) were seeded in each well of a 96-well plate in triplicate. After attachment, the cells were treated with bleomycin A5 at various concentrations (from 0 to 400 µM at 50 µM intervals) for 24, 48, or 72 h. Then cells were incubated for 3 h with CCK-8 reagent (100 µl/ml medium) (Dojindo Molecular Technologies, Inc., Kumamoto, Japan). Absorbance was determined at 490 nm using a microplate reader (Thermo Fisher Scientific Inc., Cramlington, UK).

An Annexin V/PI Apoptosis kit (Thermo Fisher Scientific Inc.) was used to assess the rate of apoptosis of fibroblast cells after bleomycin A5 treatment in accordance with the manufacturer's instructions. Briefly, after incubation in 6-well plates with DMEM/F12 with 10% FBS medium in the presence of various concentrations of BLE (0, 100, 200 or 400 µM) for 48 h, the cultured fibroblast cells were gently suspended in binding buffer and incubated for 15 min at room temperature in the dark with 5 µl Annexin V-FITC and 10 µl PI. The Annexin V-FITC- and PI-labelled cells were analyzed using a flow cytometer (BD Biosciences, Burlington, MA, USA). Using flow cytometry, dot plots of PI on the y-axis, against Annexin V-FITC on the x-axis were used to distinguish viable cells, which are negative for PI and Annexin V-FITC, cells in the early stages of apoptosis (Annexin V-positive/PI-negative), and cells in late apoptosis or full necrosis (Annexin V-FITC-positive/PI-positive staining).

After bleomycin A5 treatment at various concentrations (including 0, 100, 200, and 400 µM), fibroblasts were homogenized in 1 ml TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Total RNA was obtained, and 2 µg RNA/cell sample was reverse transcribed into complementary DNA (cDNA) using the reverse transcription system (Toyobo Co., Ltd., Osaka, Japan). Quantitative polymerase chain reaction was performed using Roche Light cycler 96 system (Roche Diagnostics, Basel, Switzerland) with a 10 µl volume mixture containing 2 µl cDNA, 0.2 µl of each primer, and 5 µl SYBR-Green (Toyobo Co., Ltd.). The relative mRNA expression levels of apoptosis-related genes were compared using the 2^−ΔΔCq method. The sequences of the primers used for PCR were ascertained from the primer bank and are listed as follows: GADPH forward, 5′-CAGTGCCAGCCTCTGCTCAT-3′ and reverse, 5′-ATACTCAGCACCAGCACAT-3′; Bcl-2 forward, 5′-CTGGGATGCCTTTGTGGAAC-3′ and reverse, 5′-GGCAGGCATGTTGACTTCAC-3′; Bax forward, 5′-CCAAGAAGCTGAGCGAGTGT-3′ and reverse, 5′-CAGCCCATGATGGTTCTGAT-3′; caspase-3 forward, 5′-ATGCAGCAAACCTCAGGGAA-3′ and reverse, 5′-GTCGGCCTCCACTGGTATTT-3′.

NPDFs treated with bleomycin A5 were homogenized in RIPA buffer (Sigma-Aldrich;. Merck KGaA, Darmstadt, Germany) on ice for 30 min. Using a BCA protein assay kit (Beyotime Institute of Biotechnology, Beijing, China), equal amounts of proteins were separated using 10% sodium dodecyl sulfate-polyacrymide (SDS) gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Milipore, Billerica, MA, USA). They were then blocked using 5% non-fat milk in TBS-0.1% Tween-20 for 1 h at room temperature. The membranes were then incubated overnight at 4°C with primary antibodies for caspase-3 (1:500 dilution), cleaved caspase-3 (1:1,000 dilution), poly(ADP-ribose) polymerase (PARP) (1:1,000 dilution), active PARP (1:1,000 dilution), Bax (1:2,000 dilution), Bcl-2 (1:1,000 dilution), or GADPH (1:10,000 dilution) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). After washing in TBST, the membranes were incubated with horseradish-peroxidase-conjugated anti-rabbit secondary antibodies for 1 h at room temperature and then visualized using an enhanced chemiluminescence kit (Millipore, Billerica, MA, USA). Images of the bands were captured using a Bio-Rad Gel Doc XR documentation system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The levels of relative protein expression were determined by densitometry and standardized to the GAPDH levels using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Mitochondria-mediated caspase-dependent pro-apoptotic protein Bax was inhibited using small interfering RNA (siRNA). Cells (2.5×10⁵/well) were plated in 6-well plates and transfected with 20 nM Bax-targeting siRNA or control siRNA (Santa Cruz Biotechnology, Inc.) using Lipofectamine 3000 kit (Life Technologies, Japan) according to the manufacturer's instructions and cultured for 72 h at 37°C in a 5% CO₂ incubator. The cells were then analyzed for Bax protein levels by western blot analysis using an anti-Bax antibody. The membranes were re-probed with an anti-GAPDH antibody to verify equal loading. Then, transfected NPDFs were treated with 200 µM BLE for 48 h and analyzed for apoptosis by Annexin V/PI staining in a flow cytometer. Annexin V⁺ cells were considered to be apoptotic cells.

All experiments were performed 3 times to provide sufficient data. The differences between the experimental conditions and the controls were analyzed through one-way analysis of variance and Tukey's test (GraphPad Prism, version 5; GraphPad Software, San Diego, CA, USA). Statistical significance was determined at the 95% confidence level. P-values <0.05 were considered to indicate a statistically significant difference.

To identify NPDFs, we first isolated fibroblast cells from nasal polyp tissue observe the cell morphologies. As showed in Fig. 1A, NPDFs were spindle and nest-like distributed. Immunocytochemistry staining of primary NPDFs indicated that the isolated and cultured cells were vimentin-positive and CK (pan)-negative, which was consistent with the characteristics of fibroblasts (Fig. 1B-D).

Next, we examined the effect of bleomycin A5 on NPDF viability. Results showed that groups treated with higher concentrations of bleomycin A5 had lower cell viability. This phenomenon was especially visible at doses ranging from 0 to 200 µM. As the duration of bleomycin A5 exposure increased, cell viability also decreased in all treated groups. In the 48 h group, the IC₅₀ = 193.0 (95% confidence intervals, 146.3 to 254.5) (Fig. 2). These results indicated that the effect of bleomycin A5 on NPDFs was concentration- and time-dependent.

Flow cytometry indicated that the ratio of apoptotic cells was 7.25±2.09% in the 0 µM group, 23.50±4.09% in 100 µM group, 55.27±8.17% in the 200 µM group, and 88.32±7.39% in 400 µM group (Fig. 3). NPDFs exposed to bleomycin A5 exhibited increased cell apoptosis in concentration-dependent manner. These results further confirmed that bleomycin A5 can induce NPDF apoptosis in a dose-dependent manner.

We next focused on identifying the pathways that lead to NPDF apoptosis using bleomycin A5. To identify the genes involved in apoptosis, we first conducted a PCR array using NPDFs treated with bleomycin A5 for 48 h at different concentrations. Using RT-qPCR, the mRNA levels of Bcl-2, Bax, and caspase-3 increased, and the difference was statistically significant (P<0.05) after bleomycin A5 treatment for 48 h (Fig. 4).

Next, we examined the changes in the protein levels of these genes using western blot analysis. Even though the 100 µM group seemed to show more caspase-3 expression than the control group, the difference was not significant. However, the levels of cleaved caspase-3 increased in a dose-dependent manner (Fig. 5). The levels of PARP, a zymolyte of cleaved caspase-3, also declined as the concentration of bleomycin A5 increased, and the level of active PARP increased in a dose-dependent manner. These results indicated that the concentrations of the apoptotic downstream effective proteins were significantly higher in both the 200 and 400 µM groups than in the control group (Fig. 5).

Bcl-2 and Bax are 2 important mitochondria-mediated caspase-dependent apoptotic proteins. It has been proven that Bax forms a complex with the anti-apoptotic protein Bcl-2 to balance its apoptotic effects (15). The ratio of Bcl-2/Bax was used to determine whether cells undergo programmed cell death. The Bcl-2/Bax ratio was determined through densitometry and normalized to the GAPDH levels of each group. The results indicated that stimulation of NPDFs with bleomycin A5 in vitro resulted in a dose-dependent decrease in the Bcl-2/Bax ratio (Fig. 6). To further confirm this conclusion, pro-apoptotic protein Bax was knockdown by siRNA transfection. The Annexin V/PI staining shows that such knockdown could significantly attenuate apoptosis in BLE-treated NPDFs (Fig. 7).