In vitro and in vivo studies of the inhibitory effects of emodin isolated from Polygonum cuspidatum on Coxsakievirus B₄

Abstract

The lack of effective therapeutics for Coxsackievirus B₄ (CVB₄) infection underscores the importance of finding novel antiviral compounds. Emodin (1,3,8-trihydroxy-6-methylanthraquinone) is one of the natural anthraquinone derivatives obtained from the root and rhizome of Polygonum cuspidatum. In the present study, the possibility of using emodin as a potential antiviral to treat CVB₄ infection was explored in vitro and in mice. Emodin reduced CVB₄ entry and replication on Hep-2 cells in a concentration- and time-dependent manner, with a 50% effective concentration (EC₅₀) of 12.06 μM and selectivity index (SI) of 5.08, respectively. The inhibitory effect of emodin for CVB₄ entry and replication was further confirmed by a quantitative real time PCR (qPCR) assay. The results further showed that the mice orally treated with different dosages of emodin displayed a dose dependent increase of survival rate, body weight and prolonged mean time of death (MTD), accompanied by significantly decreased myocardial virus titers and pathologic scores/lesions. Moreover, emodin could inhibit CVB₄-induced apoptosis in vitro and in vivo. Our results indicated that emodin could be used as potential antiviral in the post-exposure prophylaxis for CVB₄ infection.

Figure 1

Isolation and identification of emodin (a) Extraction of emodin from Polygonum cuspidatum (b) HPLC analysis for emodin standard reference (c) HPLC analysis for emodin sample isolated from Polygonum cuspidatum (d) Chemical structure of emodin.

Figure 2

Antiviral activity of emodin against CVB₄ infection. (a) MTT assay was performed to determine antiviral effect of emodin on HEp-2 cells. NC and VC indicate normal control and viral control. (b) Time-of-addition assay. HEp-2 cells were seeded into 12-well culture plates and were infected with CVB₄ for 1 h at 4 °C to allow virus binding but not virus entry, the infected cells were treated or mock treated with emodin (37 μM) or ribavirin (512 μM) or 2% test medium at the indicated times intervals of 0, 2, 4, 8, and 12 h postinfection. Virus titers were determined by plaque assay. The data are expressed as the mean ± SD of three independent experiments. ** p < 0.01 versus virus control. (c) Dose-response columns for emodin treatment in HEp-2 cells after drug incubation as determined by qPCR assay. The CVB₄ RNA levels were normalized to that of the housekeeping gene GAPDH. Each data point represents the mean ± standard deviation from three independent experiments. ** p < 0.01 versus virus control.

Figure 3

Effects of emodin on apoptosis in HEp-2 cells induced by CVB₄ infection. (a) Hep-2 cells were infected with CVB₄ and incubated with emodin (18.5, 9 μM) or ribavirin (512 μM) for 48 h, stained with Annexin-V and PI, and analyzed by flow cytometry. (b) Column bar graph of apoptosis. ** p < 0.01 versus virus control.

Figure 4

Mortality following CVB₄ infection and emodin treatment in mice. Mice challenged with 10 LD₅₀ of CVB₄ were orally administered with 30 mg/kg/d, 15 mg/kg/d, and 7.5 mg/kg/d emodin, respectively. 0.9% saline was used in viral control and normal control group. The survival rate (a), mean time to death (b), and body weight (c) of each group were determined. Statistical significance is determined by Kaplan-Meier Log Rank Test, ** p < 0.01, * p < 0.05 vs. infected control group.

Figure 5

Emodin treatment alleviates myocardial inflammation and reduced apoptosis-related genes expression in CVB₄ infected mice. (a) Mice were sacrificed at indicated times. Body weight (BW/g) and heart weight (HW/mg) of each group was measured to calculate the heart to body weight ratio (HW/BW) for each group. (b, c) Heart virus titers were determined by plaque assay on the 7th day (b) and 14th day (c) postinfection. (d) The mRNA expression of caspase-3 and bcl-2 in treated and control groups were determined by qPCR assay. ** p < 0.01, * p < 0.05 vs. infected control group.

Figure 6

Emodin treatment ameliorates CVB₄ induced heart lesions. Photomicrographs of H&E-stained paraffin sections generated from (a) normal control (b) viral control (c) ribavirin (10 mg/kg/d) (d) emodin (30 mg/kg/d) (e) emodin (15 mg/kg/d) (f) emodin (7.5 mg/kg/d). Sections from viral control displayed mononuclear cell inflammation and the appearance of multiple foci in necrotic cardiomyocytes. In groups treated with emodin or ribavirin, the lesions of the myocardium were relieved and the area of necrosis and inflammatory infiltrates was significantly decreased compared with non-treated, infected animals.

Figure 7

Detection of caspase-3 and Bcl-2 expression in emodin-treated (30, 7.5 mg/kg/d), ribavirin (10 mg/kg/d) and virus control groups by western blot analysis. GAPDH was examined to normalize any differences in loading.