Luteolin (≥98%, HPLC) was purchased from Sigma-Aldrich (Shanghai, China) and dissolved in DMSO as a stock solution. Enzyme-linked immunosorbent assay (ELISA) kits for human and mouse IFN-α and IFN-β were purchased from R&D Systems (Minneapolis, MN). Antibodies against p-STAT1Tyr701, STAT1, p-JAK1(Tyr1034/1035), JAK1, SOCS1, SOCS2, SOCS3, SOCS6, SHP-1, SHP-2 were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against CIS and GAPDH were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). A Cell Counting Kit-8 (CCK-8) and horseradish peroxidase-conjugated goat anti-rabbit antibody were purchased from Beyotime Institute of Biotechnology (Haimen, China).
Cell culture and treatment
HEp-2 and A549 cells were obtained from American Type Culture Collection (ATCC) and cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37℃ in a humidified atmosphere of 5% CO2. Human pulmonary alveolar epithelial cells (HPAEpiC) were purchased from SciencCell (Shanghai, China) and cultured in complete alveolar epithelial cell medium (AEpiCM). Murine embryonic fibroblasts (MEFs) were obtained from C57BL/6 mouse embryos (14 days) and cultured in complete DMEM. AEpiCM was purchased from ScienCell and all the other culture reagents were purchased from Gibco (Shanghai, China). Cells were pretreated with vehicle or luteolin at the indicated concentrations for 24 hours before infected with RSV at a multiplicity of infection (MOI) of 0.05. At different hours post infection (hpi), the whole cell culture was collected for plaque assay, supernatants were collected for ELISAs, or the cells were collected for RNA or protein extraction.
The long strain of RSV was obtained from ATCC and propagated in HEp-2 cells. Briefly, the virus was added to a monolayer of HEp-2 cells and allowed to absorb for 2 hours with rocking every 15 minutes. After 2 hours of absorption, the medium was replaced with DMEM containing 2% FBS. Cells were left to grow for another 4-5 days until 80-90% cytopathic effects (CPE) were observed, and then the entire cell culture was collected. After three freeze-thaw cycles, the viruses were collected by centrifugation at 50,000g for 1.5 hours at 4°C, and the remaining pellet was resuspended, aliquoted and stored at -80°C before use. Plaque forming units (PFU) were determined by plaque assays with HEp-2 cells.
To determine the EC50 (concentration for 50% of maximal effect) value of luteolin against RSV infection, A549 cells were pretreated with various concentrations of luteolin before infected with RSV. After infection for 12, 24, 48, 72 and 96 hours, the culture medium was collected for plaque assays to determine the viral titer. The viral replication inhibition rate was expressed as the percentage of viral replication compared to that in the DMSO-treated cells, and the EC50 was calculated. The selectivity indexes (SIs) for the various time points were calculated with the following equation: SI=CC50/EC50. The CC50 value (concentration of drug required to reduce cell viability by 50%) of luteolin in A549 cells was determined by CCK-8 assay.
Viral titer was determined by the plaque assay. HEp-2 cells were plated into 12-well plates and allowed to grow into a monolayer. Virus stocks, cell-free medium or lung homogenates from the mice were serially diluted 10-fold at a volume of 200 μL and then incubated with HEp-2 cells. After a 1-hour incubation, the supernatants were removed, and the cells were overlaid with 1mL of 1% (wt/vol) methylcellulose containing 50% (vol/vol) DMEM and 2% (vol/vol) FBS and cultured for another 5 days before the overlay medium was removed. Cells were fixed and stained with 2% (wt/vol) crystal violet in 20% (vol/vol) ethanol. The plaques were observed, and plaques in wells containing 30–100 plaques were counted. Viral titers were calculated using the following formula: viral titer (PFU/mL) = plaques×dilution×5.
The CC50 value of luteolin in A549 cells was determined by the CCK-8 assay. A549 cells were plated into 96-well plates at a density of 1×103/well. Twenty-four hours later, the culture media were replaced with media containing various concentrations of luteolin. After culturing for 24, 48, 72, 96 and 120 hours, 10 μL of CCK-8 solution (Beyotime Institute of Biotechnology, Haimen, China) was added, and the cells were cultured for another hour before absorbance (Ab.) at 490 nm was measured using an ELISA microplate reader (EL800, BioTek Instruments, Winooski, VT, USA). The cell inhibition rate (I%) was calculated using the following equation: I%= (Ab. control−Ab. treated)/Ab. control×100%. The CC50 value was determined.
mRNA and miRNA quantification by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis
Total cellular RNA was extracted from cultured cells or lung homogenates with TRIzol reagent (Invitrogen). To analyze mRNA expression, 1 μg of RNA was reverse transcribed to cDNA with a PrimeScript RT Reagent Kit (TaKaRa, Beijing, China), and quantitative real-time PCR was then performed with SYBR Green qPCR Master Mix (TaKaRa). Sequences of the primers used for RT-qPCR are listed in Table 1. The calculated threshold cycle was normalized based on the value of β-actin amplified from the same cDNA, and the fold-change in expression was calculated as referenced to expression of the control.
To analyze miRNA levels, total RNA was reverse transcribed into cDNA with the miRcute miRNA First-Stand cDNA Synthesis Kit (Tiangen Biotech Co., Ltd., Beijing, China). The miRcute miRNA qPCR Detection Kit (SYBR Green; Tiangen Biotech Co., Ltd.) was used for qPCR. U6 was served as the housekeeping gene. The specific primers for miR-155 and U6 were synthesized by GenePharma (Shanghai, China). The primer sequences were as follows: miR-155, forward 5′-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGAC CCCTAT-3′, reverse 5′-ACACTCCAGCTGGGTTAATGCTAATCGTGAT-3′; and U6, forward 5′-CTCGCTTCGGCAGCACA-3′, reverse, 5′-AACGCTTCACGAATTTGCG T-3′. All reactions were performed in triplicate, and the data were analyzed with the delta delta cycle threshold (CT) method of relative quantification.
To assess the production of cytokines, cell supernatants and bronchial alveolar lavage fluid (BALF) were collected. IFN-α and IFN-β levels were determined with commercial ELISA kits according to the manufacturer's instructions. The absorbance at 490 nm was read on an ELISA plate reader.
Female BALB/c mice (5-6 weeks old) were obtained from the Shanghai Laboratory Animal Company (SLAC; Shanghai, China). Current study received ethical approval from the Animal Care and Use Committee of Zhejiang University, and experiments and animal care were performed according to the approved protocols. Mice were randomly divided into three groups as follows: (i) mock-infected: mice were mock infected (DMEM only); (ii) RSV+PBS: mice were intraperitoneally injected with PBS 24 hours before RSV infection; and (iii) RSV+luteolin: mice were intraperitoneally injected with 50 mg/kg luteolin 24 hours before RSV infection. For RSV infection, mice were anesthetized and then intranasally inoculated with 5×105 or 1×107 PFU RSV in a total volume of 20 μL. At 1 or 3 days postinfection (dpi), the mice were euthanized. BALFs were harvested, and the lungs were collected for RNA extraction or fixed for hematoxylin and eosin (H&E) staining.
Western blotting was performed using the standard SDS-PAGE separation technique. The harvested cells were washed with ice-cold PBS twice and lysed in 1× RIPA buffer (Cell Signaling Technology, Danvers, MA) supplemented with 1 mM phenyl methyl sulfonyl fluoride (Solarbio, Beijing, China) and protease inhibitor cocktail. Protein concentrations were determined, and equal amounts of protein from each sample were separated by 10% SDS-PAGE and blotted onto PVDF membranes (Millipore, Billerica, MA, USA). The membranes were incubated overnight at 4°C with antibodies against p-STAT1, total STAT1, p-JAK1, total JAK1, SOCS1, SOCS2, SOCS3, SOCS6, CIS, SHP-1, SHP-2 and GAPDH. Subsequently, the membranes were incubated with a horseradish peroxidase-conjugated goat anti-rabbit antibody for 1.5 hours at room temperature. Immunoreactive protein bands were detected using an Odyssey scanning system (LI-COR, USA), quantified by ImageJ and normalized to the corresponding amount of total protein.
Luciferase reporter assay
For the IFN-stimulated response element (ISRE) luciferase assay, A549 cells in 96-well plates were transfected with plasmid (pISRE-TA-luc, Beyotime) harboring the firefly luciferase gene under control of the ISRE promoter (ISRE-luc) with or without cotransfection with SOCS1 expression plasmid (Ruijie, Shanghai, China) or a control plasmid (empty pcDNA3.1 vector) with Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Twenty-four hours after transfection, cells were treated with luteolin or vehicle for 24 hours before infection with RSV at MOI of 0.05. At the indicated hpi, cells were collected, and luciferase activities were determined with the Bright-Glo luciferase assay system (Promega Corp., Madison, WI).
The reporter constructs pcDNA-Luc Wt SOCS1 (containing the 3′-UTR of SOCS1 downstream of the luciferase gene), pcDNA-Luc Mut SOCS1 (containing a mutated target seed sequence) and Renilla vector (reference) were synthesized by GenePharma (Shanghai, China). Cells were plated in 24-well plates and then cotransfected with pcDNA-Luc Wt SOCS1 or pcDNA-Luc Mut SOCS1 and miR-155 mimic or miR-155 negative control and Renilla vector using Lipofectamine 2000. Luciferase activities were analyzed using the Dual-Luciferase® Assay system (Promega Corp., Madison, WI, USA). Relative luciferase activity was obtained by normalizing the Renilla luciferase activity to the firefly luciferase activity.
A549 cells were transfected with a SOCS1 expression plasmid or a control plasmid (empty pcDNA3.1 vector) (Ruisai Technology, Shanghai, China) with or without 50 nM hsa-miR-155 mimic or 100 nM hsa-miR-155 inhibitor (GenePharma, Shanghai, China) using Lipofectamine 2000 (Invitrogen, Shanghai, China) according to the manufacturer's instructions. Control samples were transfected with a miRNA mimic negative control (miR-155 NC). After transfection for 6 hours, cells were washed with ice-cold PBS twice and then cultured for an additional 24 hours. The transfection efficiency was assessed by RT-qPCR or Western blotting.
All data were analyzed using SPSS 17.0 software (SPSS Inc., Chicago, IL, USA). Data are presented as the means±SEM. Comparisons between two groups were performed using the Student’s t-test. P<0.05 indicated significance.