Anti-Influenza Activity of an Ethyl Acetate Fraction of a Rhus verniciflua Ethanol Extract by Neuraminidase Inhibition

Figure 5

RVSE reduced the expression of influenza A virus proteins in infected MDCK cells. The reduction of M2 proteins in MDCK cells was observed with fluorescence microscopy using the influenza A virus protein M2-specific antibodies (a). MDCK cells were also stained with DAPI (blue), and the merged images represent M2 (red). Viruses were titrated from the supernatant via the hemagglutination inhibition assay. The supernatant titer of H1N1-infected cells treated with RVSE (12.5–100 μg/mL) was significantly decreased compared with that without RVSE treatment (b, c). MDCK cells were cultured in 6-well plates ( cells/well) for 18 h. Then, H1N1 was mixed with different concentrations of RVSE (12.5, 25, 50, and 100 μg/mL), and the mixtures were incubated at 37°C for 1 h. MDCK cells were infected with these mixtures at 37°C for 2 h. Afterwards, the virus was removed, the cells were washed three times with PBS, and the medium was replaced by complete DMEM. After 8 h, the cells were harvested, and western blotting was performed using the whole cell extracts. Influenza H1N1 virus protein levels (PA, NA, NP, PB1, PB2, M1, and NS-1) in MDCK cell lysates were detected using western blotting, and β-actin was analyzed as a loading control (d, e). The blots of NA and NS-1 were stripped and reprobed using β-actin antibody. The data are representative of three independent experiments that gave similar results. Bar graph () statistics were determined by three experiments’ data using one-way ANOVA with Tukey’s post hoc test, ; . n.s.: not significant, compared with the (RVSE untreated) samples.

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