Elsevier

European Journal of Pharmacology

Volume 738, 5 September 2014, Pages 125-132
European Journal of Pharmacology

Immunopharmacology and inflammation
Antiviral activity of aloe-emodin against influenza A virus via galectin-3 up-regulation

https://doi.org/10.1016/j.ejphar.2014.05.028Get rights and content

Abstract

Novel influenza A H7N9 virus, which emerged in 2013, and highly pathogenic H5N1 virus, identified since 2003, pose challenges to public health and necessitate quest for new anti-influenza compounds. Anthraquinone derivatives like aloe-emodin, emodin and chrysophanol, reportedly exhibit antiviral activity. This study probes their inhibitory mechanism and effect against influenza A virus. Of three anthraquinone derivatives, aloe-emodin, with a lower cytotoxicity showed concentration-dependently reducing virus-induced cytopathic effect and inhibiting replication of influenza A in MDCK cells. 50% inhibitory concentration value of aloe-emodin on virus yield was less than 0.05 μg/ml. Proteomics and Western blot of MDCK cells indicated aloe-emodin up-regulating galectin-3, and thioredoxin as well as down-regulating nucleoside diphosphate kinase A. Western blot and quantitative PCR confirmed aloe-emodin up-regulating galectin-3 expression; recombinant galectin-3 augmented expression of antiviral genes IFN-β, IFN-γ, PKR and 2׳,5׳–OAS in infected cells, agreeing with expression pattern of those treated with aloe-emodin. Galectin-3 also inhibited influenza A virus replication. Proteomic analysis of treated cells indicated galectin-3 up-regulation as one anti-influenza A virus action by aloe-emodin. Since galectin-3 exhibited cytokine-like regulatory actions via JAK/STAT pathways, aloe-emodin also restored NS1-inhibited STAT1-mediated antiviral responses in transfected cells: e.g., STAT1 phosphorylation of interferon (IFN) stimulation response element (ISRE)-driven promoter, RNA-dependent protein kinase (PKR) and 2׳,5׳-oligoadenylate synthetase (2׳,5׳–OAS) expression. Treatment with aloe-emodin could control influenza infection in humans.

Introduction

Influenza A belongs to Orthomyxoviridae virus family (Wright and Webster, 2001, Nicholson et al., 2003), exhibiting 16 HA subtypes and 9 NA subtypes (Wright and Webster, 2001, Nicholson et al., 2003). H1N1, H3N2, and H1N2 subtypes cause acute respiratory disease in humans (Gamblin et al., 2004). Influenza A may mutate or re-assort with existing viruses, spawning new strains that menace public health. Swine-origin Influenza A (H1N1) virus (S-OIV), a new re-assortant strain, caused a pandemic in 2009 (Neumann et al., 2009, Schnitzler and Schnitzler, 2009). Avian influenza A viruses H5N1, H7N3, and H9N2 emerge among poultry in Asia, Africa, and Europe, occasionally causing human infection; hence our focus is on their potential for pandemic (Fauci, 2006). Avian influenza A virus H7N9 likewise caused a respiratory disease outbreak with high fatality rate in eastern China (Parry, 2013). Emerging zoonotic H7N9 and H5N1 viruses threaten public health.

Influenza A genome contains eight segmented, negative-sense single-strand RNAs encoding for hemagglutinin (HA), neuroaminidase (NA), M1, M2, nonstructural protein 1 (NS1), NP and RNP. Viral envelope spikes comprise glycoproteins HA, NA, plus M2 on the outside and M1 inside. HA homotrimer, key envelope protein forming rods, has a receptor-binding site and elicits neutralized antibodies. Its cleavage into HA1 and HA2 in acidic pH of the endosome is vital for fusion and virus infectivity (Wright and Webster, 2001, Nicholson et al., 2003). NA, a homotetramer, digests cell surface receptor (sialic acid) for release and spread of virus. M2 ion channel is responsible for endosome pH, acidifying the internal virion core to release vRNP from M1 into cell cytoplasm. NS1 protein, contributing to H5N1 avian influenza virulence, down-regulates host innate IFN-mediated antiviral response during infection (Hayman et al., 2007, Imai et al., 2010). NA and M2 inhibitors like zanamivir, oseltamivir, amantadine, and rimantadine, have been widely used to treat or prevent influenza A virus infection. Most influenza A isolates in recent years prove susceptible to neuraminidase inhibitors, but multidrug-resistant ones emerged rapidly (Ansaldi et al., 2006, Baz et al., 2007, Deyde et al., 2007), necessitating new anti-influenza compounds.

Anthraquinones like physcion, emodin, rhein, aloe-emodin, and chrysophanol isolated from Rheum palmatum and lichens, exhibit antiviral activity. Aloe-emodin possesses antiviral and anticancer potential (Barnard et al., 1992, Kubin et al., 2005, Lin et al., 2004, Mijatovic et al., 2005, Semple et al., 2001, Shuangsuo et al., 2006, Sydiskis et al., 1991), reportedly inhibiting replication of varicella-zoster, herpes simplex Types 1 and 2, pseudorabies, influenza, human cytomegalovirus, and/or Japanese encephalitis virus (Barnard et al., 1992, Sydiskis et al., 1991, Lin et al., 2008). Other anthraquinone derivatives like emodin, chrysophanic acid, and hypericin have demonstrated antiviral activity against hepatitis B/C, poliovirus, and HIV (Kubin et al., 2005, Semple et al., 2001, Shuangsuo et al., 2006). Anthraquinones directly kill enveloped viruses (Sydiskis et al., 1991). Aloe-emodin inhibits replication of un-enveloped enterovirus 71 in vitro, showing Types I and II interferon (IFN) signaling induction in mammalian cells while causing dose-dependent interferon expression and NO production (Lin et al., 2008).

This study rates antiviral effect of aloe-emodin and other anthraquinones on replication of influenza A virus in cell culture. Proteomic approach and Western blot demonstrated aloe-emodin up-regulating galectin-3 in MDCK cells. Recombinant galectin-3 showed antiviral activity, indicating galectin-3 up-regulation as involved in the antiviral mechanism of aloe-emodin against influenza. With galectin-3 proven to exhibit cytokine-like regulatory actions via JAK/STAT pathways (Jeon et al., 2010), we assessed cytokine-like regulatory effect of aloe-emodin on Type I IFN antagonistic activity of influenza A virus NS1 protein.

Section snippets

Cells and viruses

Madin-Darby canine kidney (MDCK) cells were grown as 10% Dulbecco׳s modification of Eagle׳s medium with 10% fetal bovine serum, glutamine, pyruvate and penicillin/streptomycin supplements. Influenza A/Taiwan/CMUH01/2007(H1N1) isolates were grown in MDCK cells in the same medium without serum, containing 1 μg/ml trypsin.

MTT cytotoxicity

MDCK cells were plated in 96-well plates (5×104 cells/well), then treated with serial dilution of aloe-emodin, emodin, and chrysophanol (purchased from Sigma Chemical Company).

Antiviral activity of aloe-emodin against influenza A virus

Initially, cytotoxicity of aloe-emodin, emodin and chrysophanol was rated by MTT assay (Fig. 1B): 50% cytotoxicity concentration (CC50) value of 76.6 μg/ml for aloe-emodin, 25.7 μg/ml for emodin and 18.3 μg/ml for chrysophanol to MDCK cells. To test antiviral activity of three anthraquinones, infected MDCK cells (Fig. 2A(b) were treated with (out) aloe-emodin (Fig. 2A(c), emodin (Fig. 2A(d) and chrysophanol (Fig. 2A(e) at 1 μg/ml concentration; 48 h incubation showed more than 50% rounding of

Discussion

This study documents aloe-emodin inhibiting influenza A replication and virus-induced CPE (Fig. 2, Fig. 3). Therapeutic index exceeds 10, indicating effect against influenza A; antiviral efficacy on reduction of influenza A virus-induced CPE (IC50 of 1.35±0.05 μg/ml) was more potent than that in direct virucidal assay (Wright et al., 2001). Aloe-emodin was observed directly binding with virus envelope: e.g., herpes simplex Types 1-2, varicella-zoster, pseudorabies, influenza (Baritaki and

Funding

National Science Council of R.O.C. (Taiwan), China Medical University.

Ethical approved

Not required.

Acknowledgments

We thank the National Science Council (Taiwan) and China Medical University for financial supports (NSC101-2320-B-039-036-MY3 and CMU100-ASIA-16).

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