Reagents and antibodies

Antibodies against glucose-regulated protein 78 (GRP78; 3177S), inositol-requiring enzyme 1α (IRE1α; 3294S), c-Jun N-terminal kinase (JNK; 9252S), phospho-JNK (p-JNK; Thr183/Tyr185; 4668S), p38 (8690 S), p-p38 (Thr180/Tyr182; 4511S), extracellular regulated protein kinases (ERK1/2; 4695S) and p-ERK1/2 (Thr202/Tyr204; 4370S) were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against activator protein 1 (AP-1; A11378) and sEH (A1885) were purchased from ABclonal Inc. (Wuhan, China). Antibodies against p-IRE1α (AF7150) and p-PERK (DF7576) were purchased from Affinity Biosciences (Cincinnati, OH, USA). Antibodies against PERK (24390-1-AP), activating transcription factor 6 (ATF6; 66563-1-Ig), and β-actin (66009-1-Ig), as well as the goat anti-mouse and rabbit secondary antibodies (SA00001-2, SA00001-1), were procured from Proteintech Group, Inc. (Chicago, IL, USA). Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488; ab150077) was purchased from Abcam (Cambridge, UK). Prime Script™ RT Master Mix Kit (RR036A) was procured from TaKaRa (Dalian, China). Tribromoethanol (T48402) and TRIzol reagent (T9424) were procured from Sigma-Aldrich (St. Louis, MO, USA). Lipofectamine™ RNAiMAX and bicinchoninic acid assay kits were purchased from Thermo Fisher Scientific (Waltham, MA, USA). IL-1β, monocyte chemoattractant protein-1 (MCP-1), IL-6, IL-8, and TNF-α enzyme-linked immunosorbent assay (ELISA) MAX™ Deluxe Kits were purchased from BioLegend (San Diego, CA, USA). The antifade mounting medium with 4’,6-diamidino-2-phenylindole (DAPI) (P0131) and quick genotyping assay kit for mouse tail (D7283M) were purchased from Beyotime Institute of Biotechnology (Shanghai, China). Tauroursodeoxycholate (TUDCA), KIRA6 and SP600125 were purchased from MedChemExpress (Princeton, NJ, USA).

Mice and genotypic analysis

Specific pathogen-free grade wild-type (WT) C57BL/6J mice (Beijing Huafukang Biotechnology Co., Ltd., Beijing, China) were used as the control group. sEH−/− mice (sEH−/− represents Ephx2 gene knockout) were courtesy of the Yi Zhu Laboratory [21]. WT and sEH−/− mice were maintained under standard conditions of 12-h day/night rhythm, constant temperature, and humidity levels ranging from 40 to 60%, with unrestricted access to food and water. Genomic DNA was extracted by digestion of mice tail tissues using the quick genotyping assay kit. The primers were as previously described [22]. PCR products were identified by 3% agarose gel electrophoresis, and the results were analyzed and photographed in a gel imager. A 338-base pair (bp) product was amplified for WT mice, and only a 295 bp product could be amplified for sEH−/− mice (Fig. 1A). All animal studies complied with the Animal Care and Use Committee of Capital Medical University, Beijing, China.

CS exposure

Male WT and sEH−/− mice (age: 6–8 weeks, weight: 20 ± 2 g) were randomized into four groups (n = 8) and exposed to filtered air or CS. Using a fumigation device, the WT-CS and sEH−/−-CS groups were fully exposed to 20 cigarettes (11 mg tar, 0.8 mg nicotine/cigarette; Jiangjun, Jinan, China). Mice were exposed to CS for 2 h per day, 5 days per week, for 16 weeks at a concentration of 100–120 ppm. Animal weight was measured once a week.

Lung function measurements

Mice were anesthetized with 2.5% tribromoethanol (0.1 ml/10 g, intraperitoneally). After anesthetization, mice were cannulated and connected to a flexiVent™ ventilator (SCIREQ, Montreal, Canada). We measured airway resistance (Rn), respiratory system resistance (Rrs), respiratory system compliance (Crs), tissue damping (G), tissue elasticity (H), and tissue hysteresivity (G/H).

Preparation of Bronchoalveolar Lavage Fluid (BALF)

After 16 weeks of CS exposure, all mice were anesthetized and sacrificed. The trachea was dissected for tracheal intubation, and 0.7 ml of 0.9% NaCl was withdrawn using a 1 ml syringe for lung lavage; lavage was carried out thrice. The collected fluid was BALF, centrifuged at 1,000 rpm for 10 min at 4 °C. The supernatant was collected and stored at -80 °C for subsequent measurement of inflammatory factors. BALF cell precipitates were resuspended and smeared. Neutrophils were counted by Wright-Giemsa staining.

Hematoxylin and eosin staining

Unirrigated lung tissue slices were fixed in 10% formalin for 7 days. Tissues were dehydrated in gradient ethanol and xylene, embedded in paraffin, and sliced into sections (thickness: 4 μm). Histological analysis was conducted using hematoxylin and eosin staining. The degree of emphysema was measured using the mean linear intercept (MLI) and mean alveolar number (MAN) according to previously described methodology [23].

Transmission electron microscopy

Fresh lung tissues were fixed with electron microscope fixative at 4 °C and then fixed with 1% osmium acid for 2 h at room temperature. The tissues were dehydrated in gradient alcohol for 20 min at room temperature and then in 100% acetone twice for 15 min each time. After embedding, the embedding plates were polymerized in an oven at 60 °C for 48 h. 70 nm slices were cut using an ultra-thin sectioning machine and stained in 2% uranyl acetate saturated alcohol solution and 2.6% lead citrate solution after retrieval by copper mesh. Finally, the slices were observed under a transmission electron microscope, and images were collected for analysis.

Preparation of CSE

A cigarette was continuously combusted using a peristaltic pump through 15 ml of phosphate-buffered saline in 3 min. The solution was adjusted to a pH of 7.2–7.4 with 1 M NaOH, and filtered through an aseptic 0.22 μm filter to remove insoluble particles. After adjusting the pH, the 100% CSE were aliquoted and stored at -80 °C [24].

Cell culture

Human bronchial epithelial cell line (BEAS-2B) and human peripheral blood monocytes (THP-1) were acquired from the China center for type culture collection. The BEAS-2B and THP-1 cells were cultured in DMEM/F-12 or RPMI-1640 culture medium containing 10% fetal bovine serum and 1% penicillin/streptomycin in a moistened ambient with 5% CO2 at 37 °C. The medium was replaced every 2 days. The resuscitated BEAS-2B cells were tested between the second and fifth passages.

sEH small interfering RNA (siRNA) preparation and transfection

The human Ephx2 gene was silenced using siRNA. BEAS-2B cells were seeded into 12- or 6-well plates 12 h prior to transfection. Ephx2 or negative control siRNA sequences were synthesized by RIBOBIO (Guangzhou, China). siRNA (5 nM) was transfected into BEAS-2B cells (cultured to 40–60% confluency) using Lipofectamine™ RNAiMAX Transfection Reagent according to the instructions provided by the manufacturer. Real-time quantitative PCR (qRT-PCR) analysis was used to validate Ephx2 silencing by siRNA at 48 h after transfection.

Transwell assay

BEAS-2B cells were incubated using the bottom chamber of a Transwell 24-well plate, and after completion of transfection or administration of pretreatment, 3% CSE was added to stimulate for 12 h. Seeded 200 µl of THP-1 cells in serum-free RPMI-1640 culture medium into the upper chamber. After 24 h, cells not crossing the pore size were gently wiped from the upper chamber, and the upper chamber was immersed in 4% paraformaldehyde for 20 min and stained in crystalline violet staining solution for 10 min. The staining was finished with three rinses using double distilled water. Finally, the migrating cell numbers were assessed under a light microscope at ×200 magnification.

Immunofluorescence

Lung tissue sections were deparaffinized, antigen repaired, blocked with 5% goat serum, added with primary antibodies p-IRE1α, p-PERK and ATF6, and incubated overnight at 4 °C. Similarly, cell culture dishes were fixed with 4% paraformaldehyde and sealed with 5% goat serum and then added with primary antibodies p-IRE1α, p-JNK and AP-1 for overnight incubation at 4 °C. The next day, the tissue slices and cells were incubated with fluorescent secondary antibody at 37 °C for 1 h. Next, the tissue slices and cell slides were sealed using the antifade mounting medium with DAPI. Finally, the slides were observed under a confocal laser microscope (Leica).

ELISA

The content of IL-1β and MCP-1 in serum and BALF was measured using the mouse IL-1β and MCP-1 ELISA MAX™ Deluxe kit. The concentration of IL-6, IL-8 and TNF-α in cell supernatant was assessed using the corresponding human ELISA MAX™ Deluxe kit. All operations and assays were performed according to the instructions provided by the manufacturer.

Western blot analysis

Lung tissue and cell lysates were obtained using protease and phosphoric acid protease inhibitor-containing RIPA lysates. Protein stock solutions were separated by 8% or 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. The membranes were incubated with primary antibodies against GRP78 (1:1,000), IRE1α (1:1,000), p-IRE1α (1:1,000), PERK (1:1,000), p-PERK (1:1,000), ATF6 (1:5,000), JNK (1:1,000), p-JNK (1:1,000), p38 (1:1,000), p-p38 (1:2,000), ERK1/2 (1:1,000), p-ERK1/2 (1:2,000), AP-1 (1:1,000), and β-actin (1:2,000) overnight at 4 °C with gentle shaking, then sequentially labeled with the corresponding secondary antibodies (1:5,000) for 2 h at room temperature. Blots were visualized using enhanced chemiluminescence.

Real-time quantitative PCR (qRT-PCR)

Total RNA was isolated from lung tissue and cell specimens using TRIzol reagent. Reverse transcription was carried out using the Prime Script™ RT Master Mix Kit based on the instructions provided by the manufacturer. PCR analysis was conducted using the Powe SYBR Green PCR Master Mix on the 7500 Rapid Platform. The relative mRNA expression of each group was normalized to that of β-actin, and the data were analyzed using the 2−△△CT approach. Primer sequences are summarized in Table 1.

Table 1 Primer sequences for qRT-PCR Statistical analysis

Data were analyzed using the GraphPad Prism 9 software (GraphPad Software, Inc., San Diego, CA, USA) and are presented as the mean ± SD. One-way or two-way analysis of variance was used for comparison among multiple groups. P-values < 0.05 denoted statistically significant differences.