Isochlorogenic acid C prevents enterovirus 71 infection via modulating redox homeostasis of glutathione

Figures

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  Figure 1. The molecular structure of ICAC and its effects on Vero cell viability. (A) The molecular structure of ICAC. (B) The compound was diluted to various concentrations as indicated in triplicate. The cytotoxicity of ICAC was determined by MTS assay after incubation with the drug for 48 h. The cell viability in DMEM without ICAC (control) was set as 100% (n = 6). All results were expressed as the means ± SEs. Asterisks indicate that the data significantly differ from the control group at the P < 0.05 level according to one-way analysis of variance.

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  Figure 2. The antiviral effects of IACA against EV71 in Vero cells. Vero cells were infected with 100 TCID50 of EV71 with or without different ICAC concentrations as indicated. Uninfected cells were used as the control group. (A) ICAC blocked the CPE of EV71 infection. (B) Cell viability was detected using the MTS cell proliferation assay kit at 48 hpi. The viability of the control group was set as 100% (n = 6). (C) The virions were collected by freeze-thawing at the indicated time points. The supernatant was harvested for the viral titre assay. (D) The RNA load was determined using a real-time PCR kit specific for the VP1 gene. (E) Protein samples normalized to 40 µg were subjected to 12.5% SDS-PAGE and then transferred to PVDF membrane to detect the EV71 VP1 protein expression levels. The amount of β-Tubulin was used as the internal standard. The VP1 band intensity was analysed and normalized to the corresponding band intensity of β-Tubulin. The band intensity of the EV71 group was set as 100% (n = 3). All results were expressed as the means ± SEs. Asterisks indicate that the data significantly differ from the EV71 group at the P < 0.05 level according to one-way analysis of variance.

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  Figure 3. ICAC reduced the mortality of the EV71-infected mice by regulating the viral load and cytokine secretion. Two-day-old mice were utilized as the animal model. (A) The acute toxicity of ICAC (12.5–200 mg/kg) on mice was evaluated. In the antiviral study, the mice were challenged intraperitoneally with saline (control) or 1×107 TCID50 of EV71. Then, the infected mice were treated with various doses (1.6–6.4 mg/kg) of ICAC or 10 mg/kg of ribavirin for 14 days. The mice in the model group were injected with saline. The survival rates (B) and body weight ratios (C) were recorded continuously until 14 dpi. The body weight ratio was presented relative to the corresponding treatments on the first day (n = 10). (D) Representative photos of limb paralysis caused by EV71 infection (left) and healthy phenotypes of mice treated with 6.4 mg/kg of ICAC (right) at 6 dpi. The brain tissues were sampled at 3, 6, and 9 dpi. (E) The viral loads in the brains were determined by real-time PCR (n = 6). The IL-6 (F), MCP-1 (G), and TNF-α (H) levels in the brains were determined by ELISA (n = 6). All results were expressed as the means ± SEs. Asterisks indicate that the data significantly differ from the…

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  Figure 4. ICAC maintained GSH redox homeostasis in EV71-infected Vero cells. Infected (100 TCID50 of EV71) Vero cells were treated with medium or 100 µM ICAC for 12 h. Uninfected cells were used as the control group. The reduced and oxidized glutathione (GSH and GSSG) contents were analysed by HPLC (n = 3). All results were expressed as the means ± SEs. Asterisks indicate that the data significantly differ from the EV71 group at the P < 0.05 level according to one-way analysis of variance. The GSH/GSSG ratio was also calculated.

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  Figure 5. ICAC decreased the ROS level induced by EV71 infection via modulating the antioxidant enzymes involved in GSH metabolism. Infected (100 TCID50 EV71) Vero cells were treated with medium or 100 µM ICAC for 12 h. Uninfected cells were used as the control group. The antioxidant enzymes activities (A–C) and ROS levels (D) of the Vero cells were detected (n = 3). All results were expressed as the means ± SEs. Asterisks indicate that the data significantly differ from the EV71 group at the P < 0.05 level according to one-way analysis of variance.

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  Figure 6. The antiviral effects of ICAC against EV71 were disrupted by BSO administration. Infected (100 TCID50 of EV71) Vero cells, treated with 100 µM ICAC, exposed to various concentrations of BSO (0~500 µM). Uninfected cells were used as the control group. Vero cells infected with 100 TCID50 of EV71 alone were used as the model group. Cell viability was detected using the MTS cell proliferation assay kit at 48 hpi. The viability of the control group was set as 100% (n = 6). Asterisks indicate that the data significantly differ from the ICAC group at the P < 0.05 level according to one-way analysis of variance.