Patients and nasal mucosa specimens

Patients with simple nasal septal deviation and patients with CRS with nasal polyps (hereinafter referred to as CRS) admitted in the Otolaryngology department of the First Affiliated Hospital of Guangzhou University of Chinese Medicine from April 2022 to December 2023 were selected. Patients with CRS with nasal polyps were included according to the EPOS 2020 version of the CRS diagnostic criteria [1] (specific inclusion criteria and exclusion criteria are detailed in the Additional file 1). Nasal septum mucosa or inferior turbinate mucosa of patients with simple nasal septum deviation were collected as specimens of the control group. Uncinate process mucosa were collected as representative specimens of the CRS group. All patients gave written informed consent to participate in this study, and their clinical characteristics are shown in Additional file 3: Table S1. This study was approved by the Ethics Committee of the First Affiliated Hospital of Guangzhou University of Chinese Medicine (approval No. K-2022-031).

Hematoxylin & eosin and immunofluorescence staining of tissue

Tissue specimens obtained from patients with CRS were immediately fixed in 4% paraformaldehyde and cut into coronal slices with a thickness of approximately 4 μm. The slices were stained with hematoxylin & eosin (HE) as previously described [23] and immunofluorescence (IF) was performed as previously described [14]. For IF, the slices were incubated with primary antibodies (Rabbit Anti-GSDMD-NT, AF4012, Affinity; Rabbit Anti-NLRP3, bs-10021R, Bioss; Rabbit Anti-cleaved Caspase-1, AF5418, Affinity; dilution: 1:200), then incubated with secondary antibodies (FITC labeled Goat Anti-rabbit IgG, ZF-0311, ZSGB-BIO; Alexa Fluor 594 labeled Goat Anti-rabbit IgG, ZF-0316, ZSGB-BIO; dilution: 1:50) at 24–26 ℃ in the dark for 60 min and finally stained with 4′,6-diamidino-2-phenylindole (DAPI, AR1176, Boster). The specimens were observed under a microscope (IX73, Olympus, Japan).

Cell acquisition and modelling

The uncinate process mucosa of patients with CRS was collected for culturing primary HNEpCs as previously reported [24]. HNEpCs were cultured in PneumaCultTM-Ex Basal Medium (05008, STEMCELL) with 1% penicillin- streptomycin and incubated at 37 ℃ in a cell incubator (5% CO2). The experiment was conducted using 1–3 generations of cells. HNEpCs were randomly assigned to the following groups: control, model (LPS+ATP), PLD with different concentrations (0.625, 1.25, 2.5, 5.0, 10, 20, 40, and 80 μM), MCC950 (NLRP3 inhibitor), mito-TEMPO (Mitochondrial ROS scavenger), tert-butylhydroquinone (TBHQ, Nrf2 agonist), ML385 (Nrf2 inhibitor), and ML385+PLD groups.

After 48 h of incubation, the media in the control and model groups were replaced with PneumaCultTM-Ex Basal Medium. Media in the PLD groups were replaced with media containing the corresponding concentrations of PLD (122739-11-1, Chengdu Pufei De Biotech Co., Ltd.). Media in the MCC950, Mito-TEMPO, and TBHQ groups were replaced with media containing 5 μM MCC950 (256373-96-3, MedChemExpress), Mito-TEMPO (HY-112879, MedChemExpress), and TBHQ (HY-100489, MedChemExpress), respectively. Cells were then cultured continuously for 4 h and primed with 5 μg/mL LPS (L4391-1MG, Sigma) for 10 h. Thereafter, cells were stimulated with 5 mM ATP (A6419-1G, Sigma) for 2 h to activate NLRP3 inflammasome in the model, PLD, MCC950, Mito-TEMPO, and TBHQ groups.

Media in the ML385 and ML385+PLD groups were first replaced with media containing 5 μM ML385 (HY-100523, MedChemExpress). After cells were continuously cultured for 24 h, media in the ML385 and ML385+PLD groups were replaced with media containing 5 μM ML385 and 5 μM ML385+5 μM of PLD, respectively; the cells were then continuously cultured for 4 h and primed with 5 μg/mL LPS for 10 h. Thereafter, cells were stimulated with 5 mM ATP for 2 h to activate NLRP3 inflammasome in the ML385 and ML385+PLD groups.

Cell viability assay

After treating HNEpCs as in section “Cell acquisition and modelling”, CCK-8 (CK04-500, DOJINDO) solution (1:10) was added to each well followed by incubation at 37 °C for 2 h. Then, the optical density (OD) value of each group was detected at 450 nm with a microplate reader (Multiskan GO, Thermo Scientific, USA). The cell survival rate of each group was calculated as follows: cell survival rate = (OD value of each treatment group − OD value of the background group)/(OD value of the control group − OD value of the background group) × 100%.

LDH release determination

After treating HNEpCs as in section “Cell acquisition and modelling”, the cell culture supernatant was collected as the sample to be tested. The levels of LDH in the cell culture supernatant were measured using LDH kits (A020-2-1, Nanjing Jiancheng Bioengineering Institute) according to manufacturer instructions. LDH activity (U/L) in cell culture supernatant = (OD value of each sample to be tested − OD value of the control group)/(OD value of the standard − OD value of the blank) × 0.2 × 1000.

Enzyme-linked immunosorbent assay (ELISA)

After treating HNEpCs as in section “Cell acquisition and modelling”, the cell culture supernatant was collected as the sample to be tested. The levels of IL-18 and IL-1β protein in the cell culture supernatant were measured using IL-18 ELISA kits (SEKH-0028, Solarbio) and IL-1β ELISA kits (SEKH-0002, Solarbio), respectively, according to manufacturer instructions.

Cell membrane impairment

After treating HNEpCs as in section “Cell acquisition and modelling”, the cells were washed with PBS, and then dyed with Ethidium homodimer-I (EthD-I, HR8279, Beijing Baiaolaibo) solution (1:1000) for 5 min at 37 °C. Thereafter, cores were dyed with DAPI. The specimens were observed under a microscope. The proportion of EthD-I fluorescence-positive cells = (the number of cells with both red and blue fluorescence)/(the number of blue fluorescence cells) ×100%.

Cell DNA impairment

After treating HNEpCs as in section “Cell acquisition and modelling”, the cells were washed with PBS and then fixed with 4% paraformaldehyde for 15 min. Thereafter, cells were incubated in PBS containing 0.5% Triton-X 100 for 30 min. Then, cellular DNA damage was measured using TUNEL kits (G1501, Servicebio) according to the manufacturer instructions. The specimens were observed under a microscope. The proportion of TUNEL fluorescence-positive cells = (the number of cells with both green and blue fluorescence)/(the number of blue fluorescence cells) ×100%.

Ultrastructure observation of pyroptosis cell and mitochondria

After treating HNEpCs as in section “Cell acquisition and modelling”, cells were washed with PBS and fixed with 2.5% glutaraldehyde for 2 h at 4 °C. Samples were prepared for ultrastructure observation as previously reported [14]. The images were captured using a transmission electron microscope (HITACHI HT 7800, HITACHI, Japan).

IF staining of cells

After treatment as in section “Cell acquisition and modelling”, HNEpCs were prepared for IF observation as previously described [14]. The specimens were stained with primary antibodies (Mouse Anti-GSDMD-NT, 66387-1-Ig, Proteintech; Rat Anti-NLRP3, MA5-23919, Invitrogen; dilution: 1:200) overnight at 4 °C. Subsequently, incubation with the fluorescent secondary antibody (FITC labeled Goat Anti-mouse IgG, ZF-0312, ZSGB-BIO; Alexa Fluor 594 labeled Goat Anti-rat IgG, ZF-0318, ZSGB-BIO; dilution: 1:50) was performed, followed by staining with DAPI at 37 °C. The specimens were observed under a confocal laser scanning microscope (Leica TCS SPE-II, Leica, Germany).

Reactive oxygen species (ROS) detection

After treating HNEpCs as in section “Cell acquisition and modelling”, 20 µM H2DCFDA (HY-D0940, MedChemExpress) was added to HNEpCs followed by incubation at 37 °C in the absence of light for 30 min. The cells were washed with PBS and subsequently digested with 0.25% trypsin. Following centrifugation, the cells were resuspended in PBS and immediately analyzed using flow cytometry (BD LSRFortessaTM Cell Analyzer, BD Biosciences, USA).

Mitochondrial membrane potential detection

After treating HNEpCs as in section “Cell acquisition and modelling”, 200 μM of JC-1 (HY-K0601, MedChemExpress) was directly added into the cellular medium so that the final concentration of JC-1 was 2 μM. The mixture was gently shaken and incubated at 37 °C for 20 min. After washing with PBS, red and green fluorescence was observed using a confocal laser scanning microscope. The ratio of red-to-green fluorescence was used to determine the mitochondrial membrane potential.

Western blotting (WB)

Following treatment as in section “Cell acquisition and modelling”, the HNEpCs were digested with 0.25% trypsin and collected. Protein lysis buffer was added to each sample to extract the total cellular protein. Protein concentrations were determined using the BCA method and standardized to the same concentration (3 μg/μL). The total cell protein lysate was subjected to high-temperature boiling and denaturation. Subsequently, SDS-PAGE was performed, and the resulting blots were blocked with 5% skim milk powder at 37 °C for 1 h. Blots were then exposed to primary antibodies (Rabbit Anti-NLRP3, bs-10021R, Bioss; Mouse Anti-ASC, 67494-1-Ig, Proteintech; Rabbit Anti-cleaved Caspase-1, AF5418, Affinity; Rabbit Anti-GSDMD-NT, AF4012, Affinity; Rabbit Anti-IL-18, DF6252, Affinity; Mouse Anti-IL-1β, 66737-1-Ig, Proteintech; Rabbit Anti-Nrf2, 80593-1-RR, Proteintech; Mouse Anti-HO-1, 66743-1-Ig, Proteintech; Rabbit Anti-β-actin, GB11001, Servicebio; dilution: 1:1000) and incubated overnight at 4 °C. The following day, secondary antibodies (Horseradish Enzyme-labeled Goat Anti-Rabbit IgG, ZB-2301, ZSGB-BIO; Horseradish Enzyme-labeled Goat Anti-Mouse IgG, ZB-2305, ZSGB-BIO; dilution: 1:5000) were added and incubated at 24–26 °C for 1 h. An ECL developer was added, and a gel imager (ChemiDoc MP, Bio-Rad, USA) was used for imaging analysis. The integrated optical density (IOD) of each target band was measured using the Image-J software. The IOD ratios of target proteins and β-actin were used to quantify the expression levels of the target proteins.

Statistical analysis

Image-J analysis software was used for analyzing the collected images. SPSS 24.0 software was used for statistical analysis, which was expressed as‾x ± s. All data were tested for normality and homogeneity of variance. One-way ANOVA was used for comparisons of the measurement data in each group. The LSD test was used for comparisons of homoscedasticity between groups, and Dunnett's T3 test was used for comparisons of homogeneity of variance between groups. P < 0.05 was considered statistically significant.