Mice and model establishment

In this investigation, 8-week-old female C57BL/6 N mice, obtained from Orient in Gyeongi-do, Korea, were utilized. These mice were randomly allocated into three distinct groups: control, cigarette smoking-related COPD (COPD-C), and COPD-A.

The COPD-C model was achieved by administering 80 U/kg of Porcine Pancreatic Elastase (PPE, Elastin Products Company, Owensville, MI, USA) in 50 µL of Phosphate Buffered Saline (PBS) through intratracheal (i.t.) instillation using a microsprayer aerosolizer from Penn Century Inc., located in Wyndmoor, PA, USA on day 0. Additionally, 50 µL of Cigarette Smoke Extract (CSE) was introduced intranasally (i.n.) to further mimic COPD-like conditions.

The COPD-A model was established by administering 80 U/kg of PPE in 50 µL of PBS intratracheal instillation using a micro sprayer aerosolizer on day 0. Additionally, to induce asthmatic features, 50 µg of Ovalbumin (OVA, Sigma-Aldrich, St. Louis, MO, USA) and 1 mg of aluminum hydroxide (Sigma-Aldrich) in 100 µL of PBS were injected intraperitoneally (i.p.) on days 0, 7, and 14. OVA at a concentration of 100 µg in 50 µL of PBS was intranasally given to the subjects on days 22, 23, 24, 25, 29, 32, 36, and 39, with isoflurane anesthesia provided by Vedco, located in St. Joseph, MO, USA. Additionally, to stimulate COPD features, 50 µL of CSE was also administered intranasally. Before instillation (i.t. and i.n.), mice were lightly anesthetized with isoflurane (Vedco, St. Joseph, MO, USA). Animals were humanely sacrificed on the 25th day. The experiment scheme is illustrated in Fig. 1.

For the Control group, administration of PBS (i.t) was carried out in the same manner to the PM treatment process.

Fig. 1

Scheme of (A) COPD-C and (B) COPD-A model construction and experimental workflow.COPD, chronic obstructive pulmonary disease; alum, aluminum hydroxide; CSE, cigarette smoke extract; i.n., intranasal; i.p., intraperitoneal; i.t., intratracheal; OVA, ovalbumin; PM, particulate matter; PPE, porcine pancreatic elastase

Urban PM

The Urban PM used in the study was sourced from the National Institute of Standards and Technology, specifically the SRM 1648a standard. This PM10 reference material exhibited an average particle size of 5.85 micrometers. For preparation, it underwent a dispersion process in water, utilizing ultrasonication for a duration of 10 min [23]. PM was suspended in PBS at a final concentration at 100 µg/50 µL. PM was used for treatment on days 36, 37, and 38.

Preparation of cigarette smoke extract (CSE)

Market-available cigarettes (THIS brand, 84 mm in length and 8 mm in diameter, distributed by KT&G, based in Seoul, Korea) underwent a consistent smoking process through a silicone tubing setup linked to a Variable-Flow Peristaltic Pump (manufactured by Fisherbrand in Shanghai, China). The smoke generated from a single cigarette was directed through a volume of 10 mL of PBS, at a flow rate of 50 mL per minute, sustained for a duration of 6 min [24]. Insoluble particles in the resulting suspension were filtered with a 0.22-µm filter.

Bronchoalveolar lavage fluid (BALF) collection

Bronchoalveolar lavage fluid (BALF) was collected after sacrifice under anesthetizing with an intraperitoneal injection of a mixture of Rompun and Zoletil (1:4). Methods for the BAL procedure and specimen processing were performed according to a previous experiment and are described in detail in S4.

Cytokine assay by enzyme-linked immunosorbent assay

Concentrations of neutrophil gelatinase–associated lipocalin (NGAL) in both serum and BALF and concentrations of various cytokines including interleukin (IL)-4, IL-13, IL-6, and tumor necrosis factor-α (TNF-α) in BALF were measured using ELISA kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

Lung histopathology

For scoring airway inflammation, lung tissue sample slides were numbered randomly and evaluated independently by two blinded investigators. The quantity of peribronchial or perivascular inflammation was assessed as described previously [25]. The calculation of the Mean Linear Intercept (MLI) involved the measurement of alveolar diameters across ten randomly selected fields on each slide, employing a Pannoramic MIDI slide scanner from 3DHISTECH Ltd., based in Budapest, Hungary, following previously established methods [26]. Detailed methods for lung samples processing methods were described in S6.

ROS measurement

The quantification of cellular reactive oxygen species (ROS) was carried out utilizing dihydroethidium (DHE) obtained from Invitrogen, located in Carlsbad, CA, USA. For visual documentation, each slide was imaged using the LSM 900 confocal laser scanning microscope, a product of Carl Zeiss based in Jena, Germany. Further methods for detecting ROS via DHE in lung tissue are detailed in S7.

Real-time polymerase chain reaction

Total RNAs were isolated from lung homogenates using TRIzol reagent (Invitrogen, Grand Island, NY, USA) and reverse-transcribed. Real-time PCR was performed using a CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA). Primers for PCR are listed in Table S1. The amplification process employed the iQ SYBR gene expression assay kit provided by Bio-Rad Laboratories, adhering to the guidelines specified by the manufacturer.

Western blot analysis

Antibodies against caspase-3, B-cell lymphoma protein 2 (Bcl-2), Bcl-2 associated X (Bax) from Cell Signaling Technology (Beverly, MA, USA), and transcription factor nuclear factor erythroid-derived 2-like 2 (Nrf-2) were purchased from Novous Biologicals (Centennial, CO, USA) and analyzed by Western blot. Detailed methods for Western blot analysis were described in S9.

Statistical analysis

The Data are expressed as the mean ± standard error of the mean (SEM). Group comparisons were conducted using one-way analysis of variance (ANOVA) followed by a Tukey post hoc test or two-way ANOVA coupled with the Bonferroni correction, utilizing the GraphPad Prism software (GraphPad Software, Inc., San Diego, CA, USA). Statistical significance was established at P-values of less than 0.05.