Sophocarpine against enterovirus 71 in vitro

Abstract

Sophocarpine (SCA) is a bioactive alkaloid present in Sophoraflavescens Ait. The present study demonstrated that SCA inhibited enterovirus 71 (EV71) infection in Vero cells. The results indicated that the 50% cytotoxicity concentration of SCA for Vero cells was 1,346 µg/ml, and the 50% inhibition concentration of SCA against EV71 was 350 µg/ml. SCA produced a marked inhibitory effect against EV71 when the Vero cells were treated with SCA prior to infection with the virus. Additionally, SCA was effective against EV71 when the Vero cells were infected with EV71 (100xTCID₅₀) that had been treated with SCA for 2 h, and was effective when the Vero cells were infected with EV71 (100xTCID₅₀) at 37°C under 5% CO₂ for 2 h prior to treatment with SCA for 2 h. SCA was demonstrated to inhibit the attachment and penetration of EV71 and was more effective at inhibiting attachment. The assay additionally verified that SCA suppressed the replication of viral genomic RNA and indicated that SCA may inhibit EV71 infection in vitro.

Keywords: antiviral activity; cytopathic effect; enterovirus 71; sophocarpine.

Figure 1.

After 2 days of incubation, the cytotoxicity of SCA was determined via an MTT assay. The insert in the blue box is sophocarpine structure. SCA was serially diluted to different concentrations (15.625, 31.25, 62.5, 125, 250, 500, 1,000, 2,000, and 4,000 µg/ml) in triplicate.

Figure 2.

After 2 days of incubation, the cytotoxicity of SCA was determined via an MTT assay. (A-C) Effective concentrations of SCA against EV71 were observed under an inverted microscope. (A) Vero cells. (B) EV71-infected Vero cells. (C) Vero cells exposed to 500 µg/ml SCA (magnification, ×40). (D) The Vero cells were exposed to the virus (100xTCID₅₀) and SCA. SCA was serially diluted to different concentrations (31.25, 62.5, 125, 500 and 1,000 µg/ml) in triplicate. *P<0.05 and ***P<0.001 vs. control. SCA, sophocarpine; EV71, enterovirus 71.

Figure 3.

The antiviral activity of SCA using three different treatments. (A-C) Effective concentrations of SCA against EV71 observed under an inverted microscope. (A) Vero cells, (B) EV71-infected Vero cells. (C) Vero cells were exposed to various concentrations of SCA for 2 h and then infected with EV71 (100xTCID₅₀) for 2 h (magnification, ×40). (D) Vero cells exposed to various concentrations of SCA for 2 h, then were infected with EV71 (100xTCID₅₀) for 2 h. SCA was serially diluted to different concentrations (0, 31.25, 62.5, 125, 500 and 1,000 µg/ml) in triplicate.*P<0.05 **P<0.01 and ***P<0.001 compared to the control; (E-G) Effective concentrations of SCA against EV71 observed under an inverted microscope. (E) Vero cells, (F) EV71-infected Vero cells, (G) EV71 was exposed to various concentrations of SCA prior to viral adsorption (magnification, ×40). (H) EV71 exposed to various concentrations of SCA before viral adsorption. SCA was serially diluted to different concentrations (0, 31.25, 62.5, 125, 500 and 1,000 µg/ml) in triplicate. ***P<0.001 compared to the control; (I-K) Effective concentrations of SCA against EV71 observed under an inverted microscope. (I) Vero cells, (J) EV71-infected Vero cells and (K) Vero cells were infected with EV71 (100xTCID₅₀) at 37°C under 5% CO₂ for 2 h and then supplemented with SCA (1,000 µg/ml) (magnification, ×40). (L) Vero cells were exposed to the virus (100xTCID₅₀) for 2 h and then supplemented with SCA. SCA was serially diluted to different concentrations (0, 31.25, 62.5, 125, 500 and 1,000 µg/ml) in triplicate. *P<0.05 compared to the control. SCA, sophocarpine; EV71, enterovirus 71.

Figure 4.

Effects of SCA on EV71 attachment and penetration. Images (A-C) of SCA on EV71 attachment observed under an inverted microscope. (A) Vero cells. (B) EV71-infected Vero cells. (C) Effects of SCA (1,000 µg/ml) on EV71 attachment (magnification, ×40). (D) Vero cells were infected with EV71 (100xTCID₅₀) under SCA concentrations of 0, 31.25, 62.5, 125, 500 and 1,000 µg/ml, and EV71 (100xTCID₅₀) absorption occurred for 2 h at 4°C. Images (E-G) of SCA on EV71 penetration observed under an inverted microscope. (E) Vero cells, (F) EV71-infected Vero cells. (G) Effects of SCA (1,000 µg/ml) on EV71 penetration (magnification, ×40). (H) Vero cells were infected with EV71 (100xTCID₅₀) for 3 h at 4°C to allow for EV71 attachment to the cell surface. Then, varying concentrations of SCA (1,000, 500, 250, 125, 62.5, and 31.25 µg/ml) were added. *P<0.05 and ***P<0.001 compared to the control. SCA, sophocarpine; EV71, enterovirus 71.

Figure 5.

RT-PCR products of EV71 on 1% agarose gel. The Vero cells were exposed to EV71 (100xTCID₅₀) and varying concentrations of SCA and then incubated for 48 h at 37°C. The SCA concentrations were 1,000, 500, 250, 125, 62.5, and 31.25 µg/ml, respectively.