PM (The standard reference airborne 1649b) was purchased from the National Institute of Standards and Technology (NIST, Gaithersburg, USA). PAL (powder) was obtained from Macklin (Shanghai, China). TUNEL detection kit and ROS detection kit were purchased from Beyotime (Shanghai, China). The ELISA kits of IL-18, IL-6, IL-8, SOD, MDA, and IL-1β were purchased from Boyun Biotechnology (Shanghai, China). The DMEM/F12 medium, PBS, and penicillin–streptomycin were purchased from Gibco (USA), and the fetal bovine serum (FBS) was purchased from Sijiqing (Hangzhou, China). RIPA lysis buffer and phenylmethylsulfonyl fluoride (PMSF) were purchased from Solarbio (Beijing, China) while the phosphatase inhibitors were purchased from Applygen (Beijing, China). Antibodies against Nrf2, ASC, IL-18, IL-1β, Cleaved-caspase-1, and β-actin were purchased from Abclonal (Wuhan, China); antibodies against NLRP3, HO-1, NQO1, and N-GSDMD were purchased from Affinity (Jiangsu, China); antibody against EpCAM was purchased from Santa (Cruz, USA); the secondary antibody for Western Blotting was purchased from Biosharp (Beijing, China); ML385 was purchased from MedChemExpress (Shanghai, China).

Frozen human normal lung epithelial cells Beas-2B (ATCC, USA) were removed from the liquid nitrogen tank, thawed in water at 37 ℃ until the frozen solution dissolved, and the cell suspension was transferred to a centrifuge tube and centrifuged at 1000 rpm for 3 min. Remove the supernatant, add 1 mL DMEM/F12 medium to resuspend cells, then transfer cells to culture bottle, add 4 mL DMEM/F12 medium containing 10% FBS and 1% penicillin/streptomycin, and culture in 37 ℃, 5% CO2 incubator. When the cell confluence reached 80%, the culture was subcultured to 6-well plate.

We have four groups in the cellular experiment, including the control group, PAL group, PM group, and PM + PAL group. After the confluence of cell in the 6-well plate reached 80%, the PM group and PM + PAL group were administrated with PM first; 2 h later, we gave PAL to the PM + PAL group. According to previous literature, we set concentrations of PM and PAL as 200 μg/ml and 80 μM, respectively [17, 18]. After 24 h, cells were collected for the next step.

For the cellular experiments that explored the mechanisms, we also set four groups, including the control group, PM group, PM + PAL group, and PM + PAL + ML385 group. ML385 is a specific Nrf2 inhibitor, and the dosage we give was according to previous literature [19]. After the confluence of cell in the 6-well plate reached 80%, the PM group, PM + PAL group, and PM + PAL + ML385 group were administrated with PM (200 μg/ml) first, and we gave PAL (80 μM) with or without ML385 (10 μM) to the PM + PAL group and PM + PAL + ML385 group 2 h later. After 24 h, the cells were collected for the next step.

Animal experiments were conducted using specific pathogen-free (SPF) grade C57BL/6 J male mice, aged 6–8 weeks and weighed 20–22 g, purchased from Vital River Laboratory Animal Technology Company. All animal experiments were approved by the Animal Care and Use Committee of the First Hospital of Wenzhou Medical University. After 7 days of adaptation, the mice were randomly divided into five groups: (I) control group, (II) PAL group, (III) PM group, (IV) PM + PAL (50 mg/kg) group, and (V) PM + PAL (100 mg/kg) group. According to the previous literature, we set the dosage of PM to be administered to mice at 4 mg/kg [20]. PM and PAL were both dissolved in saline. On the first day, mice were administrated orally with saline or PAL (200 μL) 2 h after intratracheal instillation with saline or PM (50 μL). On the second day, the same operation was applied to the mice. The mice were humanely euthanized 48 h after the first PM administration. Lung tissue and bronchoalveolar lavage fluid (BALF) were then collected.

Proteins of Beas-2B cells and lung tissue were extracted using RIPA lysis buffer, followed by SDS–polyacrylamide gel electrophoresis, and then transferred to nitrocellulose membranes. After incubation with primary antibody, the membranes were performed with appropriate secondary antibody. The protein bands were observed by enhanced chemiluminescence. At least three independent immunoblots were used to provide quantitative protein analysis. The gray values of protein bands were quantified by the ImageJ software.

Lung tissues were collected for histopathological examination immediately after euthanization. The lung specimens were fixed with 4% paraformaldehyde at room temperature for 24 h and then dehydrated and embedded. After dewaxing and dehydrating, the lung sections were cut to 5 μm. These lung sections were stained with hematoxylin and eosin (H&E) to assess histopathological changes. The pathological changes in the lung tissues were observed with a light microscope and evaluated on a lung injury score of 1 to 4. The pathological criteria were as follows: (1) no inflammation; (2) occasional cuffing with inflammatory cells; (3) most bronchi or vessels surrounded by a thin layer (1 to 5) of inflammatory cells; (4) most bronchi or vessels surrounded by a thick layer (> 5) of inflammatory cells [20].

The lung specimens were fixed with 4% paraformaldehyde at room temperature for 24 h. After dewaxing and dehydrating, the lung sections were cut to 5 μm. Subsequently, the lung tissue sections were stained according to the instructions. TUNEL staining of cells was also performed according to the instructions. The proportion of TUNEL-positive cells was quantified by ImageJ software.

Twenty-four hours after the cells were given the intervention, cells were washed by PBS and then incubated with DCFH-DA solution (10 µM) at 37 °C in the dark for 30 min and hoechst for 10 min. Photographs were taken with a fluorescence microscope within 30 min (excitation wavelength was 488 nm, emission wavelength was 525 nm). The mean fluorescence intensity of ROS was quantified by ImageJ software.

Lung tissues were fixed, paraffin-embedded, and sectioned. After treatment with antigen retrieval, lung sections were incubated with designated antibodies in a 4 ℃ refrigerator overnight, followed by incubation with corresponding IgG secondary antibodies, and finally stained with DAPI solution. The positive rates of NLRP3 and Nrf2 in airway epitheliums were quantified by using ImageJ software to calculate the ratio of the number of NLRP3 or Nrf2 positive cells to the EpCAM positive cells in the airway.

All data are expressed as the mean ± standard deviation (SD) of at least three independent experiments. Student’s t-test was used for comparison between different groups. For multiple comparisons, one-way analysis of variance (ANOVA) was used. Statistical analysis was performed using Prism 8 (GraphPad Prism, version 8.0.1). P < 0.05 was considered statistically significant.