Chebulagic acid, a hydrolyzable tannin, exhibited antiviral activity in vitro and in vivo against human enterovirus 71

Abstract

Human enterovirus 71 is one of the major causative agents of hand, foot and mouth disease in children under six years of age. Presently, no vaccines or antiviral drugs have been clinically available to employ against EV71. In this study, we demonstrate that treatment with chebulagic acid reduced the viral cytopathic effect on rhabdomyosarcoma cells with an IC50 of 12.5 μg/mL. The utilization of the chebulagic acid treatment on mice challenged with a lethal dose of enterovirus 71 was able to efficiently reduce mortality and relieve clinical symptoms through the inhibition of viral replication. Chebulagic acid may represent a potential therapeutic agent to control infections to enterovirus 71.

Figure 1

The structure of chebulagic acid.

Figure 2

Effect of chebulagic acid on enterovirus 71 (EV71) replication in rhabdomyosarcoma (RD) cells. (A) The infected-RD cells were treated with chebulagic acid or saline at 2 h post EV71 infection, and then the cytopathic effect (CPE) of the RD cells were observed after AO/EB double staining under a light microscope (100×) at 0 h, 48 h and 72 h post infection, respectively; (B) The viral RNA copies in the culture supernatant of the RD cells were detected by quantitative RT-PCR (qRT-PCR). The data are expressed as the mean values of three independent experiments.

Figure 3

Chebulagic acid treatment reduced the mortality of EV71-infected mice. Survival rates of the EV71-infected mice treated with the placebo, ribavirin (50 mg/kg) and chebulagic acid (0.2, 1 and 5 mg/kg) were recorded at 14 dpi (n = 20).

Figure 4

Chebulagic acid treatment relieved symptoms of EV71-infected mice. (A) The body weight of the infected mice treated with the placebo or chebulagic acid (1 mg/kg) was recorded in independent experiments (n = 20); (B) Clinical scores were systematically evaluated; (C) The typical phenotype of ruffled hair and paralysis of hind limbs caused by EV71 infection at 5 and 8 dpi (indicated by arrow) was shown, and the symptoms were prevented in the chebulagic acid treatment group (* p < 0.05, ** p < 0.01, *** p < 0.005).

Figure 5

Chebulagic acid treatment inhibited the replication of EV71 in different tissues of the mice. The infected mice were treated with the placebo or with chebulagic acid at a dose of 1 mg/kg. The tissues were sampled and subjected to viral RNA copy analysis by qRT-PCR at 6 dpi, respectively (n = 8). The data are expressed as the mean values of three independent experiments (*** p < 0.005).

Figure 6

Chebulagic acid reduced pathological damage. The infected mice were treated with the placebo or chebulagic acid at a dose of 1 mg/kg. The pathological changes of muscle tissues at 9 dpi were observed after H & E staining. (A) The necrotising myositis was observed in the placebo-treated mice (the yellow arrow); (B) Moderate inflammation was observed in the muscle tissues of chebulagic acid-treated mice (the blue arrow). Magnification: 100×.