GBD (≥98%; cat. no. 11843) and dihydroethidium (DHE; cat. no. 12013) were purchased from Cayman Chemical Company. LTA (cat. no. L2515), dimethyl sulfoxide (DMSO; cat. no. D8418), bovine serum albumin (BSA; cat. no. A5611), 2′,7′-dichlorofluorescin diacetate (DCFDA; cat. no. D6883), phenylmethylsulfonyl fluoride (PMSF; cat. no. P7626), sodium orthovanadate (cat. no. S6508), sodium pyrophosphate (cat. no. S6422), aprotinin (cat. no. A1153), leupeptin (cat. no. L2884), sodium fluoride (NaF; cat. no. 201154), and paraformaldehyde (PFA; cat. no. P6148) were purchased from MilliporeSigma. Anti-Nrf2 (cat. no. GTX103322) polyclonal antibodies (pAb) were purchased from GeneTex, Inc. Anti-HO-1 (cat. no. 10701-1-AP) pAb and β-actin (cat. no. 60008-1-Ig) monoclonal antibodies were purchased from Proteintech Group, Inc. Hybond-P polyvinylidene difluoride (PVDF) membranes (cat. no. GE10600023) and enhanced chemiluminescence Western blotting detection reagent (cat. no. GERPN2232) were obtained from Cytiva. Horseradish peroxidase (HRP)-conjugated donkey anti-rabbit immunoglobulin G (IgG; cat. no. AP182P), and sheep anti-mouse IgG (cat. no. SAB3701093) were obtained from MilliporeSigma. GBD was dissolved in DMSO, stored at 4°C, and diluted to the working concentration in cell culture medium before use. DMSO (0.1%) served as the vehicle control for all experiments.

MH-S cells, a murine alveolar macrophage cell line, were obtained from ATCC (cat. no. CRL-2019) and cultured in RPMI-1640 medium (cat. no. A1049101; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS; cat. no. 26140079; Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin. Cells were maintained at 37°C in a 5% CO₂ humidified atmosphere. Cells were used for experiments between passages 4 and 8. The cell line was confirmed to be free of mycoplasma contamination using a Mycoplasma Detection Kit (cat. no. rep-mys-10; InvivoGen).

The MH-S cells were seeded in 24-well culture plates at a density of 1×10⁵ cells per well and cultured in RPMI-1640 medium containing 10% FBS for 24 h. Once the cells reached the required confluence, they were treated with varying concentrations of GBD (10–40 µM) or 0.1% DMSO for 30 min and then subjected to LTA stimulation (10 µg/ml) for 24 h at 37°C. An MTT assay (cat. no. AM0815-0005; Bionovas Biotechnology Co., Ltd.) was performed to assesS cell viability, with results expressed as a percentage calculated using the following formula: Cell Survival Rate (%)=ODsample-ODblankODDMSO-ODblank×100%

Intracellular ROS production was assessed with DCFDA and DHE. Cells were cultured on coverslips in 6-well plates at a density of 5×10⁴ cells/well and treated with 0.1% DMSO or 20 µM GBD. The cells then underwent LTA stimulation for 30 min followed by incubation with DCFDA (10 µM) or DHE (5 µM) for 30 min at 37°C in the dark. The cells were washed twice with phosphate-buffered saline (PBS), and the coverslips were mounted onto glass slides with Fluoroshield medium containing DAPI (cat. no. ab104139; Abcam). A DM6 CS confocal microscope (Leica Microsystems GmbH) with a ×63 oil immersion objective was used for immediate fluorescence signal visualization. Fluorescence intensity was quantified using ImageJ software, version 1.54g (NIH).

Cells were cultured on coverslips at a density of 5×10⁴ per well and pretreated with 0.1% DMSO, 20 µM GBD, or 5 µM ML385 (cat. no. 21114; Cayman Chemical Company) for 30 min. The cells were subsequently stimulated with LTA for 3 or 6 h and fixed after treatment in 4% PFA for 10 min at room temperature. The fixed cells were permeabilized with 0.1% Triton X-100, followed by blocking in 5% BSA for 30 min. The prepared cells were incubated with target-specific primary antibodies (1:100) overnight at 4°C. After thorough PBS washing, the specimens were exposed to Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody (1:2,000; cat. no. ab150077; Abcam) for 1 h at room temperature. Following three additional PBS washes, the coverslips were mounted onto glass slides with Fluoroshield medium containing DAPI.

Cell imaging was performed with the aforementioned confocal microscope system. ImageJ software was used to analyze fluorescence intensity. For Nrf2 nuclear translocation, nuclear regions of interest (ROIs) were identified through DAPI staining. For HO-1 analysis, ROIs were selected around individual cells to calculate the mean fluorescence intensity (MFI) of the whole cell was calculated after background subtraction.

Cells were seeded in 6-well plates at a density of 2×10⁵ cells/well and cultured until 80% confluence. Cells were then transfected with either a small interfering (si)RNA targeting Nrf2 [Nfe2l2 siRNA: 5′-AGCAUUUUAACAUGUUAACAG-3′ (sense) and 5′-GUUAACAUGUUAAAAUGCUAU-3′ (antisense)] or a negative control siRNA [si-Ctrl: 5′-UUCUCCGAACGUGUCACGUTT-3′ (sense) and reverse 5′-ACGUGACACGUUCGGAGAATT-3′ (antisense); cat. no. HY-RS09246; MedChemExpress], using a transfection reagent (cat. no. HY-K2017, MedChemExpress) and 50 nM siRNA diluted in serum-free medium, according to the manufacturer's instructions. Following 6 h incubation at 37°C, the transfection mixture was replaced with complete medium, and cells were further cultured for 24 h before subsequent treatments. Knockdown efficiency was verified by reverse transcription-quantitative (RT-q) PCR.

Total RNA was extracted from MH-S cells (5×10⁶ cells) with a NucleoSpin RNA Kit (cat. no. 740955.50; Macherey-Nagel) according to the manufacturer's instructions. RNA purity and concentration were assessed with a NanoDrop spectrophotometer (Thermo Fisher Scientific, Inc.). Complementary DNA was synthesized from 2 µg total RNA with the SuperScript IV First-Strand Synthesis System (cat. no. 18091050; Thermo Fisher Scientific, Inc.). RT-qPCR was performed with Fast SYBR Green Master Mix (cat. no. 4385612; Applied Biosystems; Thermo Fisher Scientific, Inc.). Amplification was performed with a StepOnePlus Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.) with an initial hot-start activation at 95°C for 1 min, followed by 45 cycles of denaturation at 95°C for 5 sec and annealing/extension at 60°C for 1 min. The following primers were used: Nrf2, forward 5′-AGCAGGACATGGAGCAAGTT-3′ and reverse 5′-TTCTTTTTCCAGCGAGGAGA-3′; HO-1, forward 5′-GCACTATGTAAAGCGTCTCC-3′ and reverse 5′-GACTCTGGTCTTTGTGTTCC-3′; and GAPDH, forward 5′-GAACATCATCCCTGCATCCA-3′ and reverse 5′-GCCAGTGAGCTTCCCGTTCA-3′. GAPDH was used as the internal control. The primer sequences used for IL-1β, TNF-α, IL-6 and SOD1 are listed in Table SI. Relative gene expression levels were calculated according to the 2^−ΔΔCq method (19). All experiments were independently repeated ≥4 times.

Determination of nitric oxide and malondialdehyde levels. The detailed procedures for the determination of nitric oxide (NO) and malondialdehyde (MDA) levels are described in the Supplementary Methods.

MH-S cells (8×10⁵ per ml) were cultured and preincubated with DMSO, GBD, or ML385 for 30 min at 37°C. Cells underwent LTA stimulation or were left untreated for 3 or 6 h. The cells were lysed in lysis buffer containing aprotinin (10 µg/ml), PMSF (1 mM), leupeptin (2 µg/ml), NaF (10 mM), sodium orthovanadate (1 mM), and sodium pyrophosphate (5 mM) for 1 h at 4°C. Subsequently, the samples were centrifuged at 13,500 × g for 30 min, and protein concentration was determined through a Bradford protein assay (cat. no. 5000006; Bio-Rad Laboratories, Inc.). Equal amounts of total protein (50 µg) were separated through 10% SDS-PAGE and then transferred onto polyvinylidene difluoride membranes (MilliporeSigma). Membranes were blocked in 5% BSA in Tris-buffered saline with 0.1% Tween-20 (TBST) at room temperature for 1 h and incubated with primary antibodies (anti-Nrf2, cat. no. GTX103322, GeneTex; anti-HO-1, cat. no. 10701-1-AP and anti-β-actin, cat. no. 60008-1-Ig, Proteintech) diluted 1:1,000 in TBST overnight at 4°C. The membranes were then washed and incubated with HRP-conjugated secondary antibodies (donkey anti-rabbit IgG, cat. no. AP182P; and sheep anti-mouse IgG, cat. no. SAB3701093, MilliporeSigma) diluted 1:5,000 in TBST for 1 h at room temperature. Protein bands were quantified with a video densitometer and BioProfil BioLight software (v2000.01; Vilber Lourmat).

Cell migration was assessed through a wound healing assay performed in 24-well plates. A sterile 20-µl pipette tip was used to create a scratch in the confluent cell monolayer. Cells were incubated in serum-free medium with or without treatment. Images were captured at 0, 6, and 24 h after the scratch was created. Wound width was analyzed with ImageJ software, version 1.54g (NIH). The wound closure percentage was calculated according to the following formula: Wound closure (%)=[Wound width (0 h) - Wound width (6 h or 24 h)]Wound width (0 h)×100%

Cell migration was assessed using Transwell inserts with 8 µm pore size (SPL Life Sciences, FL, USA) in 24-well plates. MH-S cells (1×10⁵) were resuspended in medium containing 2% FBS and seeded into the upper chamber in 200 µl medium. The lower chambers were also filled with 600 µl of medium containing 2% FBS, and the plates were incubated at 37°C for 30–60 min to allow cell attachment. Cells in the upper chamber were then treated with DMSO, GBD, or ML385 for 30 min in medium containing 2% FBS at 37°C. After treatment, the medium in the lower chambers was replaced with fresh medium containing 2% FBS, with or without LTA (10 µg/ml), to serve as the chemoattractant.

After 6 or 24 h of incubation at 37°C in a humidified 5% CO₂ incubator, non-migrated cells on the upper surface of the membrane were gently removed with cotton swabs. Cells that migrated to the lower surface were fixed with 4% PFA for 15 min at room temperature, washed twice with PBS, and stained with 0.2% crystal violet for 3 min at room temperature. Excess stain was removed by washing twice with PBS. Migrated cells were imaged and counted under an inverted microscope at ×100 magnification. Relative migration was calculated as the ratio of the number of migrated cells in each treatment group to that in the DMSO group, which was defined as 1.

At least four independent replicates were performed for all experiments. Data are expressed as means±standard deviation (SD). Significance was determined through one-way analysis of variance followed by a Student-Newman-Keuls post hoc test. For datasets containing >3 groups, Tukey's post hoc test was used. P<0.05 was considered to indicate a statistically significant difference.

An MTT assay was performed to assess GBD cytotoxicity in MH-S cells. MH-S cells were treated with 10, 20 and 40 µM of GBD in the presence of LTA (10 µg/ml) for 24 h. Whereas 40 µM of GBD reduced cell viability below 80%, 10 and 20 µM had no significant cytotoxic effects (Fig. 1B). To determine the effective concentration, cells were treated with 0, 5, 10, 20, and 30 µM GBD for 24 h. Increasing concentrations of GBD reduced proliferation, with an IC₅₀ of ~18 µM (Fig. S1). Based on both of these findings, 20 µM was selected as the working concentration for subsequent experiments.

LTA stimulation markedly increased intracellular ROS production, as indicated by elevated fluorescence in both DCFDA and DHE staining (Fig. 1C and D), which suggests the oxidative environment in the MH-S cells was enhanced. Pretreatment with 20 µM GBD markedly suppressed LTA-induced ROS accumulation. Quantitative analysis revealed markedly reduced fluorescence intensity in GBD-treated cells compared with the LTA group, as measured by DHE and DCFDA staining, indicating that GBD effectively mitigated LTA-induced oxidative stress.

The present study investigated whether Nrf2 signaling contributed to the antioxidative effects of GBD. Confocal microscopy revealed that LTA stimulation alone resulted in minimal Nrf2 nuclear localization, whereas GBD pretreatment markedly increased Nrf2 nuclear accumulation (Fig. 2A).

Time-course analysis indicated that Nrf2 mRNA peaked 2 h after LTA stimulation (Fig. 2B); thus, 2 h was selected as the representative time point for mRNA analysis. Nrf2 mRNA levels were markedly elevated in the GBD+LTA group compared with in the DMSO+LTA group (Fig. 2C). Western blot analysis performed at 3 h post-LTA stimulation showed that Nrf2 protein levels were increased following GBD treatment (Fig. 2D).

HO-1 expression was further examined as a downstream target of Nrf2. Confocal imaging revealed that GBD increased HO-1 fluorescence intensity 6 h after LTA stimulation relative to LTA treatment alone (Fig. 3A). Time-course analysis revealed that HO-1 mRNA expression peaked 6 h following LTA stimulation (Fig. 3B); thus, 6 h was selected as the representative time point for subsequent analysis. GBD also markedly upregulated HO-1 mRNA and protein levels 6 h after treatment (Fig. 3C and D), confirming its downstream activation of Nrf2. HO-1 is a cytoprotective enzyme with antioxidant properties. Its upregulation by GBD may represent one mechanism through Nrf2 activation contributes to redox homeostasis. These results align with the observed decrease in ROS accumulation and support the role of HO-1 as a key effector in GBD-mediated regulation of oxidative stress.

The selective inhibitor ML385 (5 µM) was used to investigate the Nrf2-dependence of GBD-induced activation of Nrf2 and HO-1. Confocal microscopy indicated that ML385 markedly reduced the nuclear accumulation of Nrf2 in GBD-treated cells at 3 h (Fig. 4A and B). Additionally, RT-qPCR analysis revealed that ML385 suppressed GBD-induced expression of Nrf2 mRNA (Fig. 4C), indicating transcriptional inhibition.

ML385 attenuated HO-1 fluorescence intensity at 6 h, as confirmed through confocal imaging (Fig. 5A and B), and markedly decreased HO-1 mRNA levels in GBD-treated cells (Fig. 5C). These findings suggested that GBD-mediated upregulation of the Nrf2/HO-1 pathway is substantially dependent on Nrf2 activity, given that pharmacological inhibition of Nrf2 impaired both nuclear localization and downstream gene expression.

To further validate the role of Nrf2 in mediating GBD's effects, the present study performed siRNA knockdown experiments. Transfection with siRNA targeting Nrf2 (siNrf2) markedly reduced Nrf2 mRNA expression under basal conditions compared with a negative control siRNA (si-Ctrl; Fig. S2). Furthermore, Nrf2 knockdown markedly attenuated the GBD-induced upregulation of HO-1 mRNA (Fig. 5D and E). These results, together with the pharmacological inhibition by ML385, supported the involvement of Nrf2 in GBD-mediated HO-1 induction.

In addition to the Nrf2/HO-1 pathway, GBD also influenced inflammatory and oxidative stress-related factors in LTA-stimulated MH-S cells. GBD significantly decreased the mRNA expression of IL-1β, TNF-α and IL-6, while the expression of SOD1 remained unchanged at 6 h (Fig. S3). Furthermore, GBD reduced MDA and NO levels at 24 h. These findings provide additional evidence supporting the antioxidative and anti-inflammatory potential of GBD.

Wound healing assays revealed that LTA markedly promoted MH-S cell migration at 6 and 24 h. GBD pretreatment suppressed this effect at both time points (Fig. 6A and B). Notably, cotreatment with Nrf2 inhibitor ML385 attenuated the antimigratory effects of GBD, as evidenced by markedly higher closure in the ML385+GBD+LTA group than in the GBD+LTA group at 6 and 24 h. In addition, migration in ML385+GBD+LTA group remained lower than in ML385+LTA group at 6 h, although this difference was not sustained at 24 h. These findings suggested that activation of the Nrf2/HO-1 axis at least partially mediates the inhibitory effects of GBD.

To further validate the antimigratory effects of GBD, Transwell assays were performed to assess directional cell migration. LTA stimulation markedly enhanced macrophage migration at both 6 and 24 h compared with the DMSO group (Fig. 6D). GBD pretreatment markedly attenuated this increase. Consistent with Nrf2 involvement, ML385 treatment in combination with LTA promoted migration, and importantly, cotreatment with ML385 partially reversed the inhibitory effect of GBD. These results corroborated the wound-healing findings and indicated that the antimigratory effects of GBD were mediated, at least in part, by Nrf2.