Roswell Park Memorial Institute (RPMI) 1640 medium and fetal bovine serum (FBS) were from Gibco (Thermo Fisher Scientific, Inc.). Lipopolysaccharide, resveratrol, PD98059/SP600125/SB203580 (inhibitors of ERK/JNK/p38, respectively) and methyl-thiazolyl-tetrazolium (MTT) were from MilliporeSigma. Resveratrol, PD98059, SP600125 and SB203580 were dissolved in DMSO and lipopolysaccharide and MTT were diluted in a filtered PBS. Other reagents and kits included reactive oxygen species (ROS) Assay Kit (Enzo Life Sciences, Inc.), Nrf2 small interfering (si)RNA (Invitrogen; Thermo Fisher Scientific, Inc.), TRIzol^® reagent (Thermo Fisher Scientific, Inc.), total RNA isolation and reverse transcription system (Promega Corporation), mouse MUC5AC antibody (Thermo Fisher Scientific, Inc.), human heme oxygenase-1 (HO-1) antibody (Enzo Life Sciences, Inc.) and human GCLC antibody (Abnova, Inc.). Antibodies against human MUC5AC, Nrf2, β-actin, and GAPDH were purchased from Abcam. Antibodies against NAD(P)H: quinine oxidoreductase 1 (NQO1), phosphorylated (p-)ERK, ERK, p-P38, P38, p-JNK and JNK were purchased from Cell Signaling Technology, Inc.

NCI-H292 cells, a human airway epithelial cell line, was obtained from the National Collection of Authenticated Cell Cultures (Shanghai, China) and cultured in RPMI 1640 supplemented with 10% FBS +1% penicillin/streptomycin in a 5% CO₂ incubator at 37°C. When cells were ~80% confluent, varying doses of resveratrol dissolved in DMSO were added to the culture media for 30 min and then co-incubated with or without LPS (10 µg/ml) for 24 h.

The MTT assay was used to evaluate resveratrol cytotoxicity. Cells (6×10³ cells/well) were seeded in 96-well plates and treated with resveratrol (10–100 µM), with or without LPS (10 µg/ml) for 24 h. The cells were then incubated with 5 mg/ml MTT solution for 4 h and the absorbance was measured at 490 nm to estimate viable cells. The relative cell viability (%) was calculated as the ratio of the absorbance of the administered cells to that of the untreated cells.

An ROS detection kit (Enzo Life Sciences, Inc.) was used to measure the total ROS levels following the manufacturer's protocol. Briefly, cells were collected, stained with a ROS Detection Reagent and incubated in the dark at 37°C for 30 min. Finally, the fluorescence intensity of each sample was measured using flow cytometry.

NFE2L2 siRNA (Invitrogen; Thermo Fisher Scientific, Inc.) or scrambled RNA (as a negative control) was transfected into NCI-H292 cells using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Briefly, 4×10⁵ cells were seeded and cultured in 6-well plates until they reached 50–70% confluence and then treated with 50, 75, or 100 nM siRNA for 48 h at room temperature. Transfection efficiency was assessed by RT-qPCR and western blotting. The sequences were: siRNA-Nrf2 forward, 5′-AAUGAGUUCACUGUCAACUGGUUGG-3′; siRNA-Nrf2 reverse, 3′-CCAACCAGUUGACAGUGAACUCAUU-5′ and negative control (NC)-siRNA forward, 5′-UUCUCCGAACGUGUCACGUdTdT-3′ and NC-siRNA reverse, 3′-ACGUGACACGUUCGGAGAAdTdT-5′.

NCI-H292 cells were divided into six groups: siRNA-NC, LPS + siRNA-NC, LPS+ resveratrol + siRNA-NC, Nrf2-siRNA control, LPS + Nrf2-siRNA and LPS + resveratrol + Nrf2-siRNA. LPS (10 µg/ml) was added to the NCI-H292 cell culture of the LPS group for 24 h. The cells of the LPS + resveratrol group were pretreated with resveratrol (50 µM) for 30 min and then incubated with LPS (10 µg/ml) for 24 h. At 6 h after small interfering RNA transfection, the cells were incubated with LPS (10 µg/ml) or resveratrol (50 µM) + LPS (10 µg/ml) for 24 h.

Total RNA was isolated from cells (1×10⁷ cells) and lung tissues using TRIzol Reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and reverse-transcribed using a Reverse Transcription Reagent Kit (Takara Bio, Inc.) according to the manufacturer's instructions. SYBR Green Premix Ex Taq II (Takara Bio, Inc.) was used to perform real-time qPCR on an Applied Biosystems StepOnePlus system (Applied Biosystems; Thermo Fisher Scientific, Inc.). PCR primers used in this study are listed in Table I. The relative mRNA expression levels of the target genes were normalized to the CT value of GAPDH using the 2^−ΔΔCq formula (34). All the experiments were replicated three times.

Total protein was extracted using radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Institute of Biotechnology) with a protease inhibitor cocktail (Roche Diagnostics) and quantified using a BCA protein assay kit. Equal weights of proteins (20 µg/lane) were separated by SDS-PAGE on 8–10% gel and electrotransferred onto polyvinylidene difluoride membranes (MilliporeSigma). Membranes were blocked with 5% skimmed milk in tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h at 37°C and incubated with various primary antibodies overnight at 4°C. The following primary antibodies were used: Human MUC5AC (1:100, ab24070, Abcam), human Nrf2 (1:1,000, ab62352; Abcam), GAPDH (1:5,000, ab9485, Abcam), β-actin (1:5,000, ab8227, Abcam), HO-1 (1:1,000, ADI-SPA-896, Enzo Life Sciences), NQO1 (1:1,000, ma1-16672, cell signaling Technology), GCLC (1:1,000, H00002729, Cell Signaling Technology), MAPK p38 (1:1,000, #9212, Cell Signaling Technology), pp38 (1:1,000, #9215S, Cell Signaling Technology), ERK (1:1,000, #4695, Cell Signaling Technology), p-ERK (1:1,000, #4377S, Cell Signaling Technology), mouse MUC5AC (1:100, MA5-12178, Thermo Fisher Scientific). The next day, the membranes were washed in TBST and hybridized with HRP-conjugated secondary antibodies (1:10,000, BA1050 and BA1054, Wuhan Boster Biological Technology) at room temperature for 1 h. Immunoreactive protein bands were visualized using enhanced chemiluminescence (cat. no. #32109; Thermo Fisher Scientific, Inc.) on a GE ImageQuant LAS4000mini System (Cytiva). The intensities of the relative bands were quantified using Quantity One 1-D 4.62 software (Bio-Rad).

All procedures were approved by the Ethics Committee of the second affiliated hospital of Fujian Medical University (approval no. 2022-FYFE-559) and all experiments were performed in accordance with the ARRIVE guidelines for animal care (35).

A total 21 of male C57BL/6 mice (8–10 weeks old and body weight of 18–22 g) were purchased from the Experimental Animal Center of the Fujian Medical University (Fujian, China). Mice were housed under standard conditions and had unlimited access to sterilized food and distilled water. Room temperature was maintained at 25±2°C and relative humidity at 60±10% and a 12-h light/dark cycle was used. After 7 days of acclimation, the mice were randomly divided into three experimental groups (n=7 per group): i) Control group, ii) LPS exposure group and iii) LPS + resveratrol group. Group i) mice served as the normal control group and were challenged with an equal volume of PBS. Group ii) was the LPS-exposed group and the mice were treated with 100 µg LPS (in 50 µl saline) by intratracheal instillation. Group iii) mice were pretreated with an intraperitoneal injection of resveratrol (50 mg/kg) 2 h prior to intratracheal LPS instillation (100 µg/50 µl saline). The total duration of the animal study was 7 days. The mice were monitored daily for food and water intake, weight, body posture, behavior, distress and response to external stimuli. Animals were sacrificed when they reached the humane endpoints, such as a loss of >20% body weight, severe dehydration, refusal of food, severe pain or distress, or a moribund state. However, none were humanely sacrificed or found dead during the present study. All mice were anesthetized with an intraperitoneal injection of 50 mg/kg pentobarbital sodium and 0.5–1 ml blood collected by cardiac puncture, then sacrificed by exsanguination. Indexes such as breathing, heartbeat, pupils and nerve reflexes were assessed to confirm death.

The left bronchus of the mouse was ligated and ice-cold PBS (0.4 ml) was lavaged twice into the right lung. Then, the collected BALF was centrifuged at 400 × g for 10 min at 4°C and the cell pellets were resuspended in cold PBS. The total count of inflammatory cells was assessed using a chemocytometer and neutrophil counts were determined using Wright and Giemsa staining (BaSO Diagnostics Inc.).

After BALF sample collection, left lung tissue was fixed in 4% (v/v) paraformaldehyde at room temperature for 24 h, dehydrated with graded ethanol, embedded in paraffin and sliced into 4-µm sections. Hematoxylin staining was conducted at room temperature for 10 min and eosin staining for 2 min, followed by clearing in xylene for 5 min. Additionally, the slides were subjected to periodic acid Schiff (PAS) stain to assess mucus production. At room temperature, lung tissues were incubated in 0.1% periodic acid for 10 min, immersed in Schiff's reagent for 10 min, counterstained with Mayer's hematoxylin for 2 min, dehydrated in two changes of 96% alcohol, and finally cleared in xylene.

Statistical analyses were performed using the SPSS software (version 20.0; IBM Corp.) and GraphPad Prism 8.0 statistical software (Dotmatics). All data were presented as the mean ± SD. An independent-sample t-test was used to compare two groups and one-way ANOVA with Tukey's post hoc test was used to compare three or more groups of data. P<0.05 was considered to indicate a statistically significant difference.

The cytotoxicity of resveratrol on NCI-H292 cell viability was analyzed using the MTT assay at various times. As shown in Fig. 1A, ≤90 µM resveratrol and ≤70 µM resveratrol +LPS (10 µg/ml) showed no obvious toxicity to NCI-H292 cells at 24 h. Thus, concentrations of 10, 25 and 50 µM were selected for further in vitro experiments.

Previous studies, and this study, show that LPS induced MUC5AC expression in a time-dependent manner in NCI-H292 cells and that this effect was most apparent with 10 µg/ml LPS for 24 h (Fig. 1B). Therefore, for the subsequent experiments, the time point of 24 h was used. However, treatment with resveratrol significantly decreased LPS-induced MU5AC mRNA expression in a dose-dependent manner in NCI-H292 cells (Fig. 1C). Consistent with the RT-qPCR results, the expression of MUC5AC protein was markedly increased in LPS-stimulated NCI-H292 cells but was obviously suppressed in resveratrol-treated NCI-H292 cells (Fig. 1D).

To investigate the anti-oxidative effects of resveratrol, intracellular ROS production in NCI-H292 cells was measured. As shown in Fig. 2, LPS greatly induced ROS production (Fig. 2B), whereas resveratrol significantly attenuated LPS-induced ROS production (Fig. 2C).

To determine whether resveratrol inhibited LPS-induced MUC5AC expression via the Nrf2 signaling pathway, the mRNA and protein expression of Nrf2 and its downstream antioxidant genes, such as HO-1, glutamate-cysteine ligase catalytic subunit (GCLC) and NQO1 were analyzed by RT-qPCR and western blotting. First, it was found that resveratrol induced the mRNA expression of Nrf2 in a dose-dependent manner and this effect was most apparent at 50 µM (Fig. 3A). Compared with the LPS group, resveratrol significantly induced mRNA and protein expression of Nrf2 (P<0.05; Fig. 3B), as well as the downstream antioxidant genes HO-1 and GCLC (P<0.05; Fig. 3C and D). However, there was no effect on the expression of NQO1 (Fig. 3C and D).

Furthermore, the expression of Nrf2 was knocked down through siRNA transfection in NCI-H292 cells. Nrf2-siRNA could effectively decrease Nrf2 expression with 100 nM for 24 h and the mRNA and protein expression were decreased by 71.7 and 58.2% (Fig. S1). When Nrf2 expression was knocked down by Nrf2 siRNA, the downregulating effect of resveratrol on LPS-induced MUC5AC expression was significantly suppressed (P<0.05) (Fig. 3E and F). These results indicate that resveratrol inhibited LPS-induced MUC5AC expression via Nrf2 activation.

The MAPK pathway plays an important role in the regulation of inflammation, oxidative stress and mucin expression. To assess the role of the MAPK pathway in the regulatory effect of resveratrol on LPS-induced MUC5AC expression, MAPK inhibitors, including PD098059 (ERK inhibitor), SB203580 (p38 MAPK inhibitor) and SP600125 (JNK inhibitor), were used. Resveratrol failed to suppress LPS-induced MUC5AC expression in NCI-H292 cells when ERK and p38 MAPK activation were blocked by their specific inhibitors, PD098059 and SB203580 (P<0.05), respectively (Fig. 4A). However, SP600125 did not inhibit this effect. Resveratrol induced the phosphorylation of ERK and p38 MAPK (P<0.05; Fig. 4C). Additionally, the inductive effects of resveratrol on Nrf2 were suppressed by the MAPK inhibitors (Fig. 4B). These results suggested that resveratrol inhibited LPS-induced MUC5AC expression via the activation of the ERK/p38 MAPK pathway in NCI-H292 cells.

To confirm the mitigating effect of resveratrol on LPS-induced mucus hypersecretion in the lungs, the mucus expression was assessed using PAS staining, RT-qPCR and western blotting. Consistent with the in vitro experimental results, resveratrol significantly suppressed MUC5AC mRNA and protein expression in mouse lung tissues (Fig. 5A and B) and LPS-induced mucus hypersecretion (Fig. 5C).

Compared with the control group, the LPS treatment group showed a significant increase in the total number of cells and neutrophils in BALF (Fig. 6A and B). However, administration of resveratrol effectively decreased the number of inflammatory cells and neutrophils exacerbated by LPS in BALF (Fig. 6A and B). In addition, H&E staining of the lungs collected from mice in each group was performed to analyze histological changes in the lungs. Compared with LPS-challenged mice, administration of resveratrol markedly alleviated the infiltration of exacerbated inflammatory cells in the lung tissue (Fig. 6C).

The present study investigated the protein expression levels of the MAPK signaling pathway in a mouse model using western blotting. As shown in Fig. 6D, the phosphorylation levels of ERK and p38 in the resveratrol + LPS group were significantly higher than those in the LPS group. Meanwhile, the mRNA and protein level of Nrf2 were increased in the resveratrol + LPS group compared with the LPS group (P<0.05; Fig. 5A and 5).