Table 1.
Antiviral activity of S. baicalensis Georgi against CVB3 in Vero cells.
Fig 1.
The antiviral activities of norwogonin, oroxylin A, and mosloflavone against CVB3 in vitro.
(A) Antiviral activities of norwogonin, oroxylin A, and mosloflavone against CVB3 in Vero cells were measured by SRB assay. The indicated concentration of norwogonin, oroxylin A, and mosloflavone, ranging from 0.4–50 μg/ml, were added to Vero cells infected with the CCID50 titer of CVB3. Cells were cultured for 48 h and the antiviral activity was determined by CPE reduction assay. Absorbance values are presented as means ± SD from 3 independent experiments each carried out in triplicate. **P<0.001; ***P<0.0001 using one-way ANOVA with Tukey’s post hoc test. (B) The effects of norwogonin, oroxylin A, and mosloflavone on CVB3–induced CPE using SRB assay. The virus-infected cells were treated with norwogonin, oroxylin A, and mosloflavone at 50 μg/mL. After incubation at 37°C in 5% CO2 for 48 h, the morphologies of cells were photographed under a microscope. (B-a) Non-infected cells; (B-b) non-infected cells treated with norwogonin; (B-c) non-infected cells treated with oroxylin A; (B-d) non-infected cells treated with mosloflavone; (B-e) CVB3-infected cells; (B-f) CVB3-infected cells treated with norwogonin; (B-g) CVB3-infected cells treated with oroxylin A; (B-h) CVB3-infected cells treated with mosloflavone; (C) Relative CVB3 gene expression in control, CVB3-infected, and 10 μg/ml norwogonin-, oroxylin A-, and mosloflavone-treated cells by real-time PCR. **P<0.001 using one-way ANOVA with Tukey’s post hoc test. (D) TOA effects of norwogonin, oroxylin A, and mosloflavone on CVB3 replication in Vero cells. 30 μg/mL of each compound was added either during (0 h), or after (1, 2, 4, or 8 h) virus infection. After 2 days, inhibition was evaluated by the SRB method and expressed as the inhibition rate. Percentage values represent the mean ± SD of 3 independent experiments.
Table 2.
Antiviral activity of Norwogonin, Oroxylin A, and Mosloflavone against CVB3 infection in Vero cells.
Fig 2.
Oroxylin A inhibits the replication of the CVB3 replicon.
(A) Vero cells were transfected with in vitro-transcribed CVB3-replicon RNAs, immediately treated with the indicated concentrations of oroxylin A for 8h, and then assayed for firefly luciferase activity. The luciferase activity of DMSO-treated cells was considered to be 100%. **P<0.001 using one-way ANOVA with Tukey’s post hoc test. (B) In the same conditions, another set of CVB3 replicon-transfected cells was assayed for cell viability using CellTiter-Glo reagent. The activity of DMSO-treated cells was considered to be 100%.
Fig 3.
The antiviral activity of oroxylin A against CVB3 in vivo.
BALB/c mice were infected with a 1 × 106 TCID50 dose of CVB3 and given oroxylin A or vehicle. Body weights (A) and glucose levels (B) were measured for 5 days. *p < 0.05, **p < 0.01, ***p < 0.001 for CVB3 + oroxylin A versus CVB3 + vehicle, using one-way ANOVA. Virus titers of mice were determined 5 days post infection by real-time PCR (C) and western blots (D) in the pancreata. In western blot, densitometric measurement of VP1 expression is normalized to β-actin.
Fig 4.
Serum cytokine and chemokine levels in mice treated with oroxylin A.
Sera were collected on day 5 post infection and levels of IL-6 (A), CXCL1 (B), CCL2 (C), and TNF-α (D) were determined. *P<0.01;**P<0.001;***P<0.0001 using one-way ANOVA with Tukey’s post hoc test.
Fig 5.
Administration of oroxylin A prevents damage to the pancreas.
(A) Representative H&E staining of pancreas sections from uninfected mice (a), mice infected with CVB3 (b), and CVB3-infected mice treated with oroxylin A (c). (Scale bar = 20 μm) (B) Numbers of acini were counted from H&E images of pancreas sections. ***p < 0.001 as compared with the non-infected (nil) group, and #p < 0.05 for CVB3 + oroxylin A versus CVB3 + vehicle (one-way ANOVA). (C) Representative image of TUNEL staining (Green fluorescence) showing the apoptotic cells in the pancreas with red fluorescence from propidium iodide-stained nuclei. Some apoptotic cells were marked with white arrow. (D) TUNEL+ cells were counted in pancreatic sections. ***p < 0.001 (one-way ANOVA).
Fig 6.
Oroxylin A enhances eIF2α phosphorylation and NF-κB signaling.
Vero cells were infected with CVB3 and treated with oroxylin A (25 μg/ml) for 16 h. (A) Protein levels of phosphorylated eIF2α, unphosphorylated eIF2α, ATF4, and β-actin as measured by western blots. (B) The virus-infected cells were treated with salubrinal at the indicated concentrations. After incubation at 37°C in 5% CO2 for 48 h, cell viability was evaluated by SRB assay. *p < 0.05, **p < 0.01 (one-way ANOVA followed by the Tukey post hoc test).