Rheum emodin inhibits enterovirus 71 viral replication and affects the host cell cycle environment

Abstract

Human enterovirus 71 (EV71) is the primary causative agent of recent large-scale outbreaks of hand, foot, and mouth disease (HFMD) in Asia. Currently, there are no drugs available for the prevention and treatment of HFMD. In this study, we compared the anti-EV71 activities of three natural compounds, rheum emodin, artemisinin and astragaloside extracted from Chinese herbs Chinese rhubarb, Artemisia carvifolia and Astragalus, respectively, which have been traditionally used for the treatment and prevention of epidemic diseases. Human lung fibroblast cell line MRC5 was mock-infected or infected with EV71, and treated with drugs. The cytotoxicity of the drugs was detected with MTT assay. The cytopathic effects such as cell death and condensed nuclei were morphologically observed. The VP1-coding sequence required for EV71 genome replication was assayed with qRT-PCR. Viral protein expression was analyzed with Western blotting. Viral TCID50 was determined to evaluate EV71 virulence. Flow cytometry analysis of propidium iodide staining was performed to analyze the cell cycle distribution of MRC5 cells. Rheum emodin (29.6 μmol/L) effectively protected MRC5 cells from EV71-induced cytopathic effects, which resulted from the inhibiting viral replication: rheum emodin treatment decreased viral genomic levels by 5.34-fold, viral protein expression by less than 30-fold and EV71 virulence by 0.33107-fold. The fact that inhibition of rheum emodin on viral virulence was much stronger than its effects on genomic levels and viral protein expression suggested that rheum emodin inhibited viral maturation. Furthermore, rheum emodin treatment markedly diminished cell cycle arrest at S phase in MRC5 cells, which was induced by EV71 infection and favored the viral replication. In contrast, neither astragaloside (50 μmol/L) nor artemisinin (50 μmol/L) showed similar anti-EV71 activities. Among the three natural compounds tested, rheum emodin effectively suppressed EV71 viral replication, thus is a candidate anti-HFMD drug.

Figure 1

Cytotoxicity of Rheum emodin, Artemisinin and Astragaloside via MTT assay. (A) Inhibitory effects of Rheum emodin (Rheu) on MRC5 cell growth. Cells (1×10⁴ cells/well) were incubated with 0, 13.69, 27.38, 54.77, 82.15, 109.53, or 136.92 μmol/L of Rheum emodin for 48 h. (B) The inhibitory effects of Artemisinin (Arte) on MRC5 cell growth. The cells (1×10⁴ cells/well) were incubated with 0, 1, 5, 25, 50, 75, or 85 μmol/L of Artemisinin for 48 h. (C) The inhibitory effects of Astragaloside (Astra) on MRC5 cell growth. The cells (1×10⁴ cells/well) were incubated with 0, 1, 5, 25, 50, 75, or 85 μmol/L of Astragaloside for 48 h. The results indicate the mean±SD of one experiment and were representative of three independent experiments. (D) Flow diagram showing the experimental treatment protocol for MRC5 cells.

Figure 2

Effects of Rheum emodin, Artemisinin and Astragaloside on the cytopathic effects induced by EV71 infection. (A) Morphological analysis of the effects by Rheum emodin, Artemisinin or Astragaloside on cell growth. Cell growth was visualized by light microscopy at 22 h after treatment with 29.60 μmol/L of Rheum emodin, 50 μmol/L of Astragaloside or 50 μmol/L of Artemisinin. Rheu, Rheum emodin; Arte, Artemisinin; Astra, Astragaloside; Mock, mock infection; Con, 0.1% DMSO in 10% DMEM. Bar=45 μm. (B) Morphological analysis of the effect of Rheum emodin, Artemisinin or Astragaloside on cell growth after EV71 infection. Mock-infected (mock) or MRC5 cells infected with EV71 (infected) at an MOI of 1. Twenty-two hours after treatment with 29.60 μmol/L of Rheum emodin, 50 μmol/L of Astragaloside or 50 μmol/L of Artemisinin, cell growth was visualized by light microscopy. Rheu, Rheum emodin; Arte, Artemisinin; Astra, Astragaloside; Con, 0.1% DMSO in 10% DMEM. Bar=45 μm. (C) MRC5 cells were mock-infected (mock) or infected with EV71 (infected) at an MOI of 1. Twenty-two hours after treatment with 29.60 μmol/L of Rheum emodin, 50 μmol/L of Astragaloside or 50 μmol/L of Artemisinin, cells were stained with Hoechst 33258. Condensed nuclei were observed by fluorescence microscopy. Rheu, Rheum emodin; Arte, Artemisinin; Astra, Astragaloside; Con, 0.1% DMSO in 10% DMEM. Bar=15 μm. The results were from one experiment and were representative of three independent experiments.

Figure 3

Effect of Rheum emodin, Artemisinin and Astragaloside on EV71 viral genomic levels. At 12 h post-infection, intracellular EV71 RNA levels were detected in control medium (Con) or drug-containing medium-treated MRC5 cells by real-time quantitative PCR. (A) 29.60 μmol/L of Rheum emodin (Rheu) treatment for 10 h. The results are standardized using GAPDH RNA as a control and normalized to 100 in control cells. (B) 50 μmol/L of Artemisinin (Arte) for 10 h. The results are standardized using GAPDH RNA as a control and normalized to 1.0 in control cells. (C) 50 μmol/L of Astragaloside (Astra) treatment for 10 h. The results are standardized using GAPDH RNA as a control and normalized to 1.0 in control cells (C). The results indicate the mean±SD of one experiment and were representative of three independent experiments. ^*P<0.05, ^**P<0.01.

Figure 4

Western blotting shows the effects of Rheum emodin, Artemisinin and Astragaloside on EV71 protein expression. VP1 expression was determined after growth in control medium (–) or drug medium (+) at 26 h and 36 h post-infection. S, supernatant protein; C, intracellular protein; T, supernatant and intracellular protein. Rheu, Rheum emodin; Arte, Artemisinin; Astra, Astragaloside; Con, 0.1% DMSO in 10% DMEM. The numerical value is the ratio of VP1 protein intensity in the control group to that in the drug treatment group. Data were representative of three independent experiments.

Figure 5A

Effect of Rheum emodin, Artemisinin and Astragaloside on the cell cycle manipulation of EV71 infection. Cell cycle profiles were determined by flow cytometry after drug treatment or EV71 infection (A).

Figure 5B–5G

Effect of Rheum emodin, Artemisinin and Astragaloside on the cell cycle manipulation of EV71 infection. Histograms were analyzed by the ModFit LT program to determine the percentage of cells in each phase of the cell cycle at 24 h post-infection in the mock-infected (Mock) and EV71-infected (infected) MRC5 cells in (B, D, and F). Control medium (Con) and 29.60 μmol/L of Rheum emodin (Rheu) treatment for 22 h (B). Control medium (Con) and 50 μmol/L of Artemisinin (Arte) treatment for 22 h (D). Control medium (Con) and 50 μmol/L of Astragaloside (Astra) treatment for 22 h (F). The S phase percentage was compared between the control medium (Con) and the drug treatment groups in the EV71-infected (Infected) MRC5 cells in (C, E, and G). Control medium (Con) and 29.60 μmol/L of Rheum emodin treatment (Rheu) for 10 h (C). Control medium (Con) and 50 μmol/L of Artemisinin (Arte) treatment for 10 h (E). Control medium (Con) and 50 μmol/L of Astragaloside (Astra) treatment for 10 h (G). The results indicate the mean±SD of one experiment and are representative of three independent experiments. ^**P<0.01.

Figure 6

Western blotting shows the effects of Rheum emodin, Artemisinin and Astragaloside on expression of host cell cycle regulatory proteins after EV71 infection. MRC5 cells were mock-infected or infected with EV71 at a MOI of 1 for 2 h. Two hours later, the cells were treated with drugs that lasted for 34 h, and then the cells were collected for lysis. Cyclin A2 and CDK2 expression was detected by Western blot analysis. Control medium and 29.60 μmol/L of Rheum emodin (Rheu) treatment for 34 h (A). Control medium and 50 μmol/L of Artemisinin (Arte) treatment for 34 h (B). Control medium and 50 μmol/L of Astragaloside (Astra) treatment for 34 h (C). Histone or Tubulin was shown as a loading control. The results are representative of three independent experiments.