Virus and cells
Cell lines of 16HBE (human bronchial epithelial cells), BEAS-2B (human bronchial epithelial transformed cell) and Vero cells (African green monkey kidney cells) were obtained from ATCC. HEp-2 (laryngeal squamous cell carcinoma cells) and A549 (lung adenocarcinoma cells) cells were obtained from Prof. Enmei Liu（Children’s Hospital of Chongqing Medical University）. The cells were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (AusGeneX).
All RSV infections were carried out by RSV A2 strain, which was obtained from Prof. Enmei Liu（Children’s Hospital of Chongqing Medical University）. The RSV A2 strain was prepared in HEp-2 cells as previously described . Briefly，HEp-2 cells inoculated with RSV A2 were incubated with DMEM containing 2% FBS at 37 °C (5% CO2) until the cells exhibited >90% cytopathic effect (CPE). The cultures were then frozen and thawed, and the cell lysates containing viral particles were collected and centrifuged at 4 °C，at 12000 rpm for 10 minutes to remove the cell debris. The viral stocks were titrated by agarose plaque assay, and then stored at -80℃ in small aliquots.
In STS treatment assays, virus at a multiplicity of infection (MOI) of 0.01 was added to 80% confluent cells and left to adhere at 37℃ for 2h，followed by washing twice with PBS to remove unabsorbed virus. Then the cells were cultured in DMEM supplemented with different concentration of sodium tanshinone IIA sulfonate (STS, MedChem Express, New Jersey, USA) (12.5，25，50，100 and 200μg/ml or only 25μg/ml ) [17, 19, 21] and 2% FBS, cells or supernatants samples were collected at 12h, 24h, 36h,48h or 60h post RSV infection. In STS efficiency assay (percent of inhibition or promotion), virus infection was similar, except the cells were covered with the mixture consisted with 1% agar and 2×DMEM supplemented with 5% FBS containing different concentration of STS (0-200μg/ml). STS efficiency was defined as the ratio of plaques formed on treated versus untreated cells.
Supernatants of RSV infected cells were collected, and tittered by plaque assay on HEp-2 monolayers with neutral red staining. Viral titers were calculated as plaque-forming units (PFU) as previously described.
Cell Viability tests
Cell viability testing was done by using a Cell Counting Kit-8 (CCK8) according to the manufacturer’s instructions. Briefly, 1×10^5 A549 cells were seeded in 96-well plates. They were left to adhere overnight and then treated with STS (0-200μg/ml) for 48 h, then the supernatants were removed and cells were washed with PBS for three times. CCK8 reagent was added to the wells for 1 h at 37 °C. Then the OD values were measured at 450nm with a microplate reader and cell viability was calculated.
Cells were grown in 24-well plates to about 80% confluence, then infected with RSV and treated with STS as described above. The infected cells were washed three times with PBS and fixed in 4% formaldehyde for 20 min, followed by 1% BSA (Solarbio, Beijing, China) with 0.3% Triton X-100 (Solarbio, Beijing, China) in PBS treatment for 30min. Cells were then stained with a mouse monoclonal antibody against RSV N (1:200, mouse; Abcam, Cambridge, UK) in 1% BSA at 37℃ for 2h. Cells were washed with PBS for three times, then FITC 488-conjugated goat anti-mouse secondary antibody (1:300; Bioss, China) were incubated at 37℃ for 1h. Images were obtained using a Olympus inverted immunofluorescence microscope.
The levels of IFN-α, IFN-β and IP-10 in supernatants of infected cells were measured with commercial ELISA kits of IFN-α, IFN-β and IP-10 (Neobioscience, Wuhan, China) according to the manufacturer’s instructions.
Western blots analysis
Cells in 6-well plates were washed twice with PBS, then protein was extracted using a total protein extraction kit (KeyGEN, Nanjing, China). After protein quantification using a BCA assay reagent (Solarbio, Beijing, China), equal amounts of protein (60 μg) were separated on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and then transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA). The membranes were blocked and then incubated with a mouse antibody against RSV N (1:1000, mouse; Abcam, Cambridge, UK) or GAPDH (1:5,000, rabbit; Bioss, China) at 4°C overnight. Then, an HRP-conjugated goat anti-mouse secondary antibody (1:5,000; Bioss, China) or a goat anti-rabbit secondary antibody (1:5,000; Bioss, China) were used to detect the presence of the respective protein bands. Signals were quantified using the Quantity One software (Bio-Rad, Hercules, CA) and normalized relative to GAPDH.
The GraphPad Prism 5.0 software was used for data analysis. All results are expressed as the mean ± SEM unless otherwise stated. Statistical significance was analyzed by a student’s t-test and ANOVA. Differences were considered significant at P < 0.05.
The study was approved by the Ethics Committees of the China Guizhou Provincial People’s Hospital (Approve number 2018058).