V2O5 (pure 98%, 181.88 MW, CAS #: 1314-62-1) and silibinin (pure 98%, 482.44 MW, CAS #: 22888-70-6) were purchased from Sigma Aldrich. Gefitinib and imatinib (pure 100%, 589.7 MW, CAS #: 13166, 13139) were purchased from Cayman Chemical (Ann Arbor, MI, USA). 6-shogaol was purchased from Chengdu Biopurify (PS010913, Chengdu Push Biotechnology Co, Ltd, China). This research was confirmed that all experiments were performed in accordance with ARRIVE guidelines.
The human lung embryo cell line, L132, was purchased from the Korea Cell Line Bank (Seoul, Korea). Cells were maintained at 37°C in a humidified atmosphere containing 5% CO2 in RPMI 1640 supplemented with 10% fetal bovine serum (Gibco) and 1% antibiotics.
Cell viability assay.
Cell were seeded into 96-well plates (1 × 103 cells/well) and allowed to attach overnight. They were then treated with various concentrations of compounds or dimethyl sulfoxide for 0, 24, 48, 72, and 96 h. Cells were treated with various concentrations (1, 2, 5, 10, and 15 µM) of V2O5 and incubated at 37°C for 1 h. Subsequently, 10 µl CCK-8 reagent was added to each well and the cells were incubated for an additional 2 h. The absorbance was measured at 450 nm using a microplate reader. When the compounds was evaluated, the cells were treated in the same manner. The compounds were used at a concentration of 20 µM 1 h before V2O5 treatment.
Eight-week-old male BALB/c mice were maintained in an environment a humidity of 50 ± 10%, a 12 h light/dark cycle, and a temperature of 22 ± 2°C. The mice were provided with tap water and weighed weekly. The Animal Testing Ethics Committee of Kyungpook National University approved this study for animal experiments (approval no. 2019-0056).
Establishment of lung injury mice.
Mice were randomized in to four groups (6 mice per group): (1) control; (2) V2O5; (3) V2O5 + silibinin 50 mg/kg; (4) V2O5 + silibinin 100 mg/kg. Silibinin was dissolved in 100 µl of distilled water at a dose of 50 mg/kg (low dose) and 100 mg/kg (high dose). Mice were pretreated with silibinin 50 mg/kg; 100 mg/kg in 100 µl by oral administration, 1 h before V2O5 exposure. The mice were placed in a whole-body inhalation chamber (Gaon bio, Yongin, Republic of Korea), where they were exposed to particulate aerosols of V2O5 concentration of either 0 (control) or 4 mg/m3 (V2O5 group, silibinin 50 mg/kg group, silibinin 100 mg/kg group) for 6 h per day, 3 days per week for 8 weeks 20,47. The mice were sacrificed after the final exposure by cervical dislocation.
Whole blood analysis.
Immediately after sacrifice, whole blood was obtained from the mice and analysed using an ADVIA 120 Hematology system (Korea Polytech College, Nonsan, Korea). Next, cell number analysis of white blood cells (WBC), neutrophils, lymphocytes, and eosinophils were performed.
Mouse lung tissues were fixed with 4% formaldehyde, paraffin-embedded, and cut into 4-µm sections. The sections were stained with hematoxylin–eosin (H&E). Each section was observed using a light microscope (Olympus BX43, Olympus, Tokyo, Japan) to estimate inflammatory cell infiltration. Furthermore, the bronchial thickness in lung tissue and H scoring was determined by skilled researchers. The H score was considered 5 points if the intensity of inflammation was severe and 1 point if inflammation was weak.
RNA extraction and real-time PCR.
Total RNA was isolated from lung tissues using TRIzol reagent. Total RNA was converted to cDNA using the PrimeScript™ 1st strand cDNA synthesis kit (6110, Takara, China). For PCR amplification, the following primers were used: mouse ꞵ-actin forward, 5’-GGC TCT TTT CCA GCC TTC CT-3’ and reverse, 5’-GTC TTT ACG GAT GTC AAC GTC ACA-3’; IL-1ꞵ forward, 5’-CCC CAG GGC ATG TTA AGG A-3’, and reverse, 5’-TGA CCC TGA GCG ACC TGT CT-3’; IL-6 forward, 5’-GTT GTG CAA TGG CAA TTC TGA-3’, and reverse, 5’-TTG GTA GCA TCC ATC ATT TCT TTG-3’; TNF-α forward, 5’-AGG ACC CAG TGT GGG AAG CT-3’, and reverse, 5’-AAA GAG Prime Script GCA ACA AGG TAG AGA-3’. The PCR reaction mixture contained 8 µl cDNA, 10 µl Power SYBR Green PCR Master Mix (4367659, Applied Biosystems, UK), 1 µl 0.2 pmol forward primer, and 1 µl 0.2 pmol reverse primer. The Applied Biosystems real-time PCR program consisted of a holding stage at 95°C for 10 min, followed by 40 cycles of cycling at 95°C for 15 s, 60°C for 1 min, followed by a melt curve. The relative expression of IL-1ꞵ, IL-6, and TNF-α mRNA were normalized to that of ꞵ-actin mRNA.
Western blot analysis.
Lung tissues were homogenized in tissue lysis buffer (Intron Biotechnology, Korea). Protein concentration was determined using the BCA Protein Assay reagent (Thermo Scientific) according to the manufacturer’s instructions. Equal amounts of protein lysate was separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were incubated overnight with primary antibodies at 4°C. The following primary antibodies and dilutions were used: TNF-α, IL-1ꞵ, phospho-p38, total p38, phospho-JNK, total JNK, phospho-ERK1/2, total ERK1/2, phosphor-p65, total p65, NLRP3 (1:1000 dilution; Cell signaling Technology, MA, USA), SOD1, and SOD2 (1:1000 dilution; Santacruz Biotechnology, CA, USA). Subsequently, the membranes were incubated with corresponding secondary antibodies for 1 h at room temperature. Immunoblots were visualized using ECL detection kit (GE Healthcare, Seoul, Korea) by Imagequant™ LAS 500 (GE Healthcare).
Immunohistochemical analysis (IHC).
NLRP3 proteins in lung tissues were examined with immunohistochemical (IHC) staining. The samples were fixed with paraformaldehyde at 4°T for 4 h, washed with phosphate-buffered saline containing 20% sucrose for 4 h, embedded and cut into 4-µm-thick sections on acid pretreated slides. After dewaxing, blocking endogenous peroxidase, and repairing the antigen, the lung tissue sections were incubated with anti-rabbit NLRP3 antibodies (1:200 dilution) at 4°C overnight, followed by incubation with HRP-labeled Goat Anti-Mouse IgG (H + L) as a secondary antibody (1:100 dilution) at 37°C for 30 min. The results were observed under a light microscope.
All results are expressed as the means ± standard deviation (SD) from at least three independent experiments. All analyses were performed using SPSS software (IBM SPSS Statistics 26.0). Normality tests were performed with Kolmogorov-Smirnov and Shapiro-Wilk test (P > 0.05). Statistical significance between experimental groups was determined using one‑way ANOVA for pair‑wise comparisons with Dunnett’s test. P < 0.05 was considered to indicate a statistically significant difference.