A549 cells were subjected to GSPE pretreatment and infected with RSV. The experimental group underwent GSPE pretreatment and was infected with RSV; the negative (infected) control was not GSPE-pretreated. Another cell group received GSPE pretreatment, but was not infected. Uninfected cells with no pretreatment served as the reference. Fold changes in gene expression were calculated, relative to the reference group. Samples were taken after 24, 48 and 72 h of culture. All experiments were repeated three times. The experimental design is presented in Fig. 1.

GSPE from Vitis vinifera was supplied by Hanlim Pharmaceutical Co., Ltd. (CAS no. 71328-22-8; Seoul, Korea). GSPE contains proanthocyanidins (80% of all solids) and some catechin monomers (12). GSPE was solubilized in PBS. All chemicals were of analytical grade and were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany) unless otherwise indicated. Dulbecco's modified Eagle's medium (DMEM), penicillin-streptomycin and trypsin-EDTA were purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). RPMI-1640 was purchased from Corning Inc. (Corning, NY, USA) and fetal bovine serum (FBS) was obtained from GE Healthcare (Chicago, IL, USA). R&D Systems, Inc. (Minneapolis, MN, USA) supplied the ELISA kits used to measure IL-1β, IL-6 and IL-8 expression. A primary antibody against IL-1β (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and an appropriate secondary antibody (Cell Signaling Technology, Inc., Beverly, MA, USA) were used for western blotting.

The A549 human lung adenocarcinoma epithelial cell line and HeLa derivative HEp-2 cells were purchased from the Korean Cell Line Bank (Korean Cell Line Research Foundation, Seoul, South Korea). A549 cells were cultured in RPMI-1640 medium supplemented with 10% (v/v) FBS, 100 U/ml penicillin, 100 µg/ml streptomycin and 2 mM glutamine. Cells were maintained in a 37°C humidified incubator under 5% CO₂ and 95% air (both v/v) until the desired confluency (80%) was attained.

RSV strain A2 was obtained from the American Type Culture Collection (Manassas, VA, USA). The virus was grown in HEp-2 cell monolayers with DMEM and 10% (v/v) FBS, and purified as previously described (13,14). Viral titer and growth were determined by quantitative plaque assays (15).

A549 cells at ~80% confluence were infected with RSV at a multiplicity of infection (MOI) of 3 for 60 min at 37°C. Following adsorption, the viral solution was removed, cells were rinsed twice with PBS, and incubation was continued in RPMI-1640 medium supplemented with 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin and 2 mM glutamine for 72 h at 37°C. Samples were taken at 24, 48 and 72 h for RNA and protein extraction; the culture supernatants were stored at −80°C prior to ELISAs. Cells infected with RSV served as negative controls and uninfected cells as the reference group.

The potential effects of GSPE on A549 cell viability were determined. Cells were grown in 6-well plates (1×10⁵ per well) for 24 h at 37°C, following which GSPE was added at the indicated concentrations (5, 10, 20, 30, 40, 50 and 100 µg/ml); the control group received 0.01% (v/v) PBS. After incubation at 37°C for 24, 48 and 72 h, cells were harvested with trypsin-EDTA solution (JBI, Seoul, South Korea), washed once in PBS with 5% (v/v) FBS, and counted using an ADAM-MC cell counter (NanoEnTek, Inc., Seoul, South Korea).

The mRNA expression of IL-1β, IL-6 and IL-8 was measured by RT-qPCR. Total cellular RNA was obtained with TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). First-strand cDNA was synthesized by reverse transcription in a reaction mixture (20 µl) containing 1 mM of each dNTP, 1 µg RNA, 1X reaction buffer, 5 µM random primers and 20 units AMV reverse transcriptase (Promega Corporation, Madison, WI, USA). cDNA synthesis was performed at 25°C for 10 min for primer annealing, 42°C for 60 min for reverse transcription and 95°C for 5 min for transcriptase denaturation. The sequences of the gene-specific primers used were as follows: IL-1β forward, 5′-TGATGGCTTATTACAGTGGCAATG-3′ and reverse, 5′-GTAGTGGTGGTCGGAGATTCG-3′ (140 bp); IL-6 forward, 5′-GTCTTGCCTGCTGCCTTC-3′ and reverse, 5′-AGTGCCTCTTTGCTGCTTTC-3′ (194 bp); IL-8 forward, 5′-GACATACTCCAAACCTTTCCAC-3′ and reverse, 5′-CTTCTCCACAAACCTCTGC-3′ (160 bp); and β-actin forward, 5′-GCGAGAAGATGACCCAGATC-3′ and reverse, 5′-GGATAGCACAGCCTGGATAG-3′ (77 bp). PCR was performed in a StepOnePlusÔ Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). Each reaction mixture (20 µl) contained 1 µl cDNA, 1 µl solutions of both the reverse and forward primers, 10 µl Power SYBR-Green PCR Master Mix, and 7 µl PCR-grade water. The amplification protocol included initial denaturation at 95°C for 10 min, followed by 40 cycles of denaturation (95°C for 15 sec) and annealing (60°C for 1 min). Relative gene expression was calculated using the 2^−∆∆Cq method (16). The data were normalized to the control group and are presented as fold changes in mRNA expression (three experiments/group).

A549 cells were seeded into 6-well plates at a density of 1.5×10⁵ cells/well and incubated in RPMI-1640 with 2% (v/v) FBS for 72 h at 37°C to avoid the confounding effect of serum-induced cytokine expression. Cells were cultured (with or without GSPE pretreatment) for 24 h prior to RSV infection (at a MOI of 3) for 1 h at 37°C. After 24, 48 and 72 h of infection, the culture media were collected and centrifuged at 2,000 × g for 10 min at 4°C to remove cell debris. The supernatants were subjected to IL-1β (cat. no. DLB50), IL-6 (cat. no. D6050) and IL-8 (cat. no. D8000C) (all from R&D Systems, Inc.) ELISAs, in accordance with the manufacturer's instructions.

Pretreated A549 cells were infected with RSV (at a MOI of 3) in serum-free RPMI-1640 for 60 min at 37°C, and grown under optimal conditions for 72 h at 37°C. Next, cells were washed with cold PBS and lysed with lysis buffer (Cell Signaling Technology, Inc.). A bicinchoninic acid protein assay was used to determine total protein concentration. Protein (30 µg) was mixed with loading buffer, boiled for 5 min and loaded onto 12% (w/v) polyacrylamide gels. After electrophoresis, proteins were transferred onto polyvinylidene difluoride membranes. Membranes were blocked in 5% (w/v) non-fat milk powder solution for 1 h at room temperature, and subsequently incubated overnight at 4°C in Tris-buffered saline with 0.05% (v/v) Tween-20 (TBS-T) containing primary antibodies against IL-1β (1:250; cat. no. sc-7884; Santa Cruz Biotechnology, Inc.) and β-actin (1:1,000; cat. no. 4970S; Cell Signaling Technology, Inc.). Following washing with TBS-T, the membranes were soaked in TBS-T containing goat anti-rabbit IgG horseradish peroxidase-conjugated secondary antibodies (1:2,500; cat. no. 7074; Cell Signaling Technology, Inc.) for 1 h at room temperature. Following a further wash with TBS-T, target protein bands were visualized using an Enhanced Chemiluminescence kit (Thermo Fisher Scientific, Inc.) and a Davinch-Chemi™ Chemiluminescence Imaging system (Davinch-K Co., Ltd., Seoul, South Korea). Densitometric analysis of scanned immunoblot images was performed using ImageJ software version 1.49 (National Institutes of Health, Bethesda, MD, USA).

All values are expressed as the mean ± standard deviation. SPSS version 23.0 (IBM Corp., Armonk, NY, USA) was used for statistical analyses. SigmaPlot version 10 (Systat Software, Inc., Chicago, IL, USA) was used for plotting graphs. Two-way analysis of variance followed by Bonferroni's post-hoc test was used to determine the statistical significance of differences between GSPE pretreatment groups and groups subjected to RSV infection alone. In all analyses, P<0.05 was considered to indicate a statistically significant difference.

The optimal concentration for GSPE treatment was evaluated by exposing A549 cells to varying concentrations (5, 10, 20, 30, 40, 50 and 100 µg/ml) of GSPE for 24, 48 and 72 h. The data are expressed as cell numbers compared with those in the control group (Fig. 2). It was determined that GSPE was not toxic at either 5 or 10 µg/ml; therefore, these concentrations used in all subsequent experiments.

To evaluate the effects of GSPE pretreatment on RSV-infected A549 cells, the mRNA expression of IL-1β, IL-6 and IL-8 was determined by RT-qPCR. RSV infection significantly increased the mRNA expression of IL-1β, IL-6 and IL-8, compared with the control group. However, GPSE pretreatment significantly decreased the expression of IL-1β mRNA (from 24.23- to 0.59-fold at 5 µg/ml, and to 0.20-fold at 10 µg/ml after 48 h; Fig. 3A). In addition, the mRNA expression of IL-6 significantly decreased (from 939.04- to 6.13-fold at 5 µg/ml, and to 0.80-fold at 10 µg/ml after 48 h; Fig. 3B). Similarly, the expression of IL-8 mRNA decreased (from 120.85- to 3.87-fold at 5 µg/ml, and to 0.84-fold at 10 µg/ml; Fig. 3C). All changes were significant compared with the RSV infection-alone group.

ELISAs were performed to confirm the effects of GSPE on proinflammatory cytokine protein expression. However, the ELISA did not detect IL-1β protein expression; thus additional western blot analysis was performed, using a polyclonal antibody against IL-1β. Negligible IL-1β expression was detected at baseline, but expression increased upon RSV infection (at an MOI of 3). GSPE pretreatment (5 or 10 µg/ml) significantly reduced IL-1β expression (Fig. 4). The levels of IL-6 (Fig. 5A) and IL-8 (Fig. 5B), as determined by ELISAs, significantly increased upon RSV infection (at an MOI of 3) compared with the control, whereas GSPE pretreatment significantly reduced the production of these factors.