Antiviral Mechanisms of Saucerneol from Saururus chinensis against Enterovirus A71, Coxsackievirus A16, and Coxsackievirus B3: Role of Mitochondrial ROS and the STING/TKB-1/IRF3 Pathway

Figures

• 

  Figure 1. Antiviral activity of saucerneol against enterovirus A71 (EV71) in vitro. (A) Vero cells were infected with 5 × 103 PFU EV71 and treated with saucerneol. Cell viability and cytotoxicity by saucerneol treatment were measured using the sulforhodamine B (SRB) assay. (B) Relative EV71 5′ noncoding region (NCR) gene expression in EV71-infected Vero cells was determined using real-time PCR. (C) Western blot analysis of VP3 in EV71-infected cells after treatment with the vehicle or 0.4–50 µg/mL of saucerneol for 24 h. The ratio of VP3 to β-actin based on the quantification of bands in the immunoblot. Rupintrivir was used as a positive control. For the validation assessment, (A) were obtained through three independent experiments and (B,C) were obtained through two independent experiments. The results are shown as the mean ± SEM. ** p < 0.01 and *** p < 0.001 for comparison with the EV71-infected vehicle-treated group (Veh) based on one-way ANOVA with Bonferroni’s multiple comparison test.

• 

  Figure 2. Antiviral effect of saucerneol confirmed using immunofluorescence. (A) Representative image of VP3 staining in EV71-infected and non-infected Vero cells treated with 10 µg/mL saucerneol for 24 h. The image was observed under a confocal microscope. Red signals represent VP3; blue signals represent DAPI-stained nuclei. Scale bar, 50 µm. (B) MFI values of immunofluorescence images were quantified using ZEN software. The data were obtained through two independent experiments for validation assessment. The results are shown as mean ± SEM. *** p < 0.001 for comparison with the EV71-infected vehicle-treated group (Veh) based on one-way ANOVA with Bonferroni’s multiple comparison test.

• 

  Figure 3. Saucerneol inhibited EV71 and coxsackievirus A16 (CVA16) replication at the early stage of viral infection. (A) Vero cells were treated with 10 µg/mL saucerneol during or after infection with EV71 at the indicated time points and viral mRNA was analyzed 14 h post infection. (B) Vero cells were treated with 10 µg/mL saucerneol during or after infection with CVA16 at the indicated time points and viral mRNA was analyzed 14 h post infection. (C) Vero cells were infected with EV71 and harvested at the indicated time points, including at 4, 6, 8, 10, and 12 h after treatment with 10 µg/mL saucerneol. (D) Vero cells were infected with 1 × 103 PFU CVA16 and harvested at the indicated time points, including at 4, 6, 8, 10, and 12 h after treatment with 10 µg/mL saucerneol. Rupintrivir was used as a positive control. The data were obtained through two independent experiments for validation assessment. Total RNA was isolated and viral RNA was analyzed using reverse transcription–real-time PCR.

• 

  Figure 4. Saucerneol induced mitochondrial ROS. (A) EV71-infected Vero cells were treated with 10 µg/mL saucerneol. After 2 h of infection and treatment, harvested cells were stained with 5 µM of Mito-SOX reagent and analyzed using fluorescence-activated cell sorting (FACS) to detect reduction in mitochondrial ROS. The mean Mito-SOX fluorescence index (MFI) was measured. (B) EV71-infected Vero cells were treated with 10 µg/mL saucerneol. After 4 h of infection and treatment, harvested cells were stained with 5 µM of Mito-SOX reagent and analyzed by fluorescence-activated cell sorting (FACS) to detect any reduction in mitochondrial ROS. The Mito-SOX MFI was measured. (C) Mito-TEMPO (50 µM) was added to saucerneol-treated and EV71-infected cells. For the validation assessment, (A,B) were obtained through two independent experiments and (C) were obtained through three independent experiments. The results are shown as mean ± SEM. * p < 0.05, ** p < 0.01 and ns (not significant) for comparison with each group based on one-way ANOVA with Bonferroni’s multiple comparison test.

• 

  Figure 5. Saucerneol induced the STING/TBK-1/IRF3 signaling pathway in Enterovirus 71- and Coxsackievirus A16-infected cells. (A) Representative immunoblot results of STING, p-STING, TBK-1, p-TBK-1, IRF3, p-IRF3, and β-actin to evaluate activation of their expression in (A) EV71 and (B) CVA16 -infected Vero cells or uninfected cells after treatment with saucerneol (10 µg/mL). The samples were derived from the same experiment, and gels/blots were processed in parallel.

• 

  Figure 6. Antiviral activity of saucerneol against coxsackievirus B3 (CVB3) in vivo. (A) BALB/c mice were infected intraperitoneally with a 1 × 106 PFU of CVB3 and then assessed by body weight. Saucerneol treatment was administered intraperitoneally at 2 mg/kg daily until the mice were euthanized 8 days post infection. (B) Enterovirus RNA levels in mice pancreas treated with saucerneol. RNA was isolated and viral RNA was analyzed using reverse transcription–real-time PCR. (C) Representative hematoxylin and eosin (H&E)-stained images of pancreas sections showing uninfected, CVB3-infected, and CVB3-infected groups treated with saucerneol (scale bar = 20 μm). The data were obtained through two independent experiments for validation assessment. ** p < 0.01 for comparison with the CVB3-infected group based on one-way ANOVA with Bonferroni’s multiple comparison test.