Artemisia capillaris inhibited enterovirus 71-induced cell injury by preventing viral internalization

Abstract

Artemisia capillaris (A. capillaris) is a common herbal drug used for thousands years in ancient China. A. capillaris has been empirically used to manage hand-foot-mouth disease (HFMD), which is commonly caused by enterovirus 71 (EV71). EV71 can cause meningoencephalitis with mortality and neurologic sequelae without effective management. It is presently unknown whether A. capillaris is effective against EV71 infection. To test the hypothesis that it could protect cells from EV71-induced injury, a hot water extract of A. capillaris was tested in human foreskin fibroblast cells (CCFS-1/KMC) and human rhabdomyosarcoma cells (RD cells) by plaque reduction assay and flow cytometry. Inhibition of viral replication was examined by reverse quantitative RT-PCR (qRT-PCR). Its effect on translations of viral proteins (VP0, VP1, VP2, protease 2B and 3AB), and apoptotic proteins were examined by western blot. A. capillaris was dose-dependently effective against EV71 infection in both CCFS-1/KMC cells and RD cells by inhibiting viral internalization. However, A. capillaris was minimally effective on viral attachment, VP2 translation, and inhibition of virus-induced apoptosis. Further isolation of effective molecules is needed. In conclusion, A. capillaris has anti-EV71 activity mainly by inhibiting viral internalization. A. capillaris would be better to manage EV71 infection in combination with other agents.

Keywords: Alternative therapy; Gan-Lu-Siao-Du-Yin; TCM; Traditional Chinese medicine.

Figure 1

The HPLC‐UV chromatographic fingerprint profile of Artemisia capillaris (AC). (A) Standard reference of chlorogenic acid. (B) The hot water extract of A. capillaris (H₂O‐AC). Peak No. 6 was chlorogenic acid. (C) The methanol extract of A. capillaris (MeOH‐AC). Peak No. 6 was chlorogenic acid.

Figure 2

Cytotoxicity and antiviral effect of A. capillaries. The infection rate and viability of CCFS‐1/KMC cells determined by plaque assay and XTT test respectively (a), and the infection rate of RD cells estimated by flow cytometric assay (b) are shown. Thirty μg/mL ribavirin served as a positive control for antiviral assay. UV‐inactivated EV71 served as a blank control. Data are represented as mean ± SD of 9 tests in plaque reduction assay and of 4 tests in flow cytometric assay. *p < 0.05; **p < 0.001; ***p < 0.0001 were compared to the negative control (0 μg/mL; EV71).

Figure 3

Time‐dependent anti‐EV71 activity of A. capillaries. The infection rates of CCFS‐1/KMC cells treated with various concentrations of A. capillaris at 1 h or 2 h prior to or after infection as indicated are shown. Data are presented as mean ± SD of 9 tests. *p < 0.05; **p < 0.001; ***p < 0.0001 were compared to the viral control (0 μg/mL).

Figure 4

Effect of A. capillaris on viral attachment and internalization. The infection rates of CCFS‐1/KMC cells treated with various concentrations of A. capillaris were determined by the attachment assay (a) and internalization assay (b). Data are presented as mean ± S.D. of 9 tests. *p < 0.05; **p < 0.001; ***p < 0.0001 were compared to the viral control (0 μg/mL).

Figure 5

Effect of A. capillaris on EV71 genome levels determined by qRT‐PCR are shown. 30 μg/mL ribavirin served as a positive control. UV‐inactivated EV71 served as a blank control. Please note the different unit of intracellular viral RNA and viral RNA in the suspension. Data are represented as mean ± S.D. of 3 tests relative to UV‐EV71 group. *p < 0.05; **p < 0.001; ***p < 0.0001 were compared to the viral control group (0 μg/mL).

Figure 6

Effect of A. capillaris on the protein translations determined by western blot are shown. 30 μg/mL ribavirin served as a positive control. UV‐inactivated EV71 served as a blank control.