May 10, 2014 Antiviral activity of aloe-emodin against influenza A virus via galectin-3 up-regulation
Shih-Wen Li1 Tsuey-Ching Yang2, Chien-Chen Lai3 Su-Hua Huang4 Jun-Ming Liao1 Lei Wan5 Ying-Ju Lin5 Cheng-Wen Lin 1,4*
1Department of Medical Laboratory Science and Biotechnology, China Medical University, Taichung 404, Taiwan
2Department of Biotechnology and Laboratory Science in Medicine, National Yang Ming University, Taipei, Taiwan
3Institute of Molecular Biology, National Chung Hsing University, Taichung, 402 Taiwan
4Department of Biotechnology and Bioinformatics, Asia University, Taichung 413, Taiwan
5Department of Medical Genetics and Medical Research, China Medical University Hospital, Taichung, 404 Taiwan
Co-first author.
*Corresponding author.
Mailing address: Department of Medical Laboratory Science and Biotechnology, China Medical University, No. 91, Hsueh-Shih Road, Taichung 404, Taiwan Phone: 886-4-22053366 ext. 7210 Fax: 886-4-22057414. Email: [email protected] 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
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. A 50% inhibitory concentration value of aloe-emodin on virus yield was less 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.
Keywords: Influenza A virus, anthraquinone, aloe-emodin, galectin-3 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54
1. 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 et al., 2009). Avian influenza A viruses H5N1, H7N3, and H9N2 emerge among poultry in Asia, Africa, and Europe, occasionally causing human infection; hence our focus 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 antiviralresponse during infection (Hayman et al., 2007; Imai et al., 2010). NA and M2 inhibitors like 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79
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; Abed and Boivin, 2007; Deyde et al., 2007), necessitating new anti-influenza compounds.
Anthraquinones like physcion, emodin, rhein, aloe-emodin, chrysophanol, isolated from Rheum palmatum and lichens, exhibit antiviral activity. Aloe-emodin possesses antiviral and anticancer potential (Barnard et a., 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), 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104
we assessed cytokine-like regulatory effect of aloe-emodin on Type I IFN antagonistic activity of influenza A virus NS1 protein.
2. Materials and Methods 2.1. Cells and viruses
Madin-Darby canine kidney (MDCK) cells were grown 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.
2.2. 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). After 48-h incubation at 37℃ in 5% CO2, 25 l of MTT
solution at 5 mg/ml was added to each well and reacted for 3 h. After three phosphate buffer saline washings, 100 l DMSO was added to plates for dissolving formazan crystals. OD570-630 in each well was tested by micro-ELISA reader, survival rate (%) indexing MDCK suppression by each compound: ((Acontrol−Aexperiment)/Acontrol) × 100%. Cytotoxic concentration giving 50% (CC50) was calculated by computer program (provided by John Spouge, NCBI, NIH).
2.3. Viral yield assay using real-time RT-PCR
To quantify antiviral activity of aloe-emodin, emodin, and chrysophanol, MDCK cells (1 × 107) were infected with Influenza A (MOI=1) and simultaneously treated with or without each compound. After 48-h infection, cultured supernatant was 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128
collected; viral genomes were extracted by QIAamp Viral RNA Mini Kit (Qiagen). Real-time RT-PCR used specific primers, SYBR greenPCR Master Mix and SYBR green I dsDNA bindingdye. Oligonucleotide primers for M gene were forward 5’-AAGACCAATCCTGTCACCTCTGA-3’ and reverse 5’-CAAAGCGTCTACGCT GCAGTCC -3’. PCR product level was monitored by ABI PRISM 7000 sequence detection system (Applied Biosystems).
2.4. Plaque reduction assay
To test viral plaque reduction effect, aloe-emodin (0 μg/ml, 0.1 μg/ml, 1 μg/ml, 2.5 μg/ml) was added, along with influenza A at 100 pfu, into the well of MDCK cell monolayer at 37 °C for 1 h, then overlaid with MEM medium containing 0.3% BSA, 0.9% agar, and 1 μg/ml trypsin. After 3-day incubation, plaques were stained with 0.1% crystal violet solution containing 10% formaldehyde. Data represent means ±
S.D. of three independent experiments. Inhibitory concentration showing 50% plaque reduction (IC50) was calculated by computer. Inhibitive rate gauged aloe-emodin
effect on plaque assay: inhibition (%) = ((Plaque＃of mock control–Plaque＃of aloe-emodin treatment)/ Plaque＃of mock control)) × 100%, with 50% inhibitory
concentration (IC50) derived by computer.
2.5. Two-dimensional gel electrophoresis and protein spot identification
Two-dimensional gel electrophoresis, in-gel digestion of protein spots and nanoelectrospray mass spectrometry proceeded as described earlier (Kubin et al., 2005; Shuangsuo et al., 2006). Lysate (100 μg) from mock or treated cells was diluted with 350 μl of rehydration buffer and applied to nonlinear Immobiline DryStrips, then applied for first-dimensional isoelectric focus with Multiphor II system and second-dimension electrophoresis by 12% polyacrylamide gels. After staining with silver nitrate solution, gels were scanned by GS-800 imaging densitometer with PDQuest 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153
software version 7.1.1 (Bio-Rad). After trypsin digestion of excised spots, digested peptides were separated by RP C18 capillary column, then electrosprayed into Q-TOF mass spectrometer. Protein identification was based on assignment of at least two peptides, protein function and subcellular location annotated by Swiss-Prot (http://us. Expasy.org/sprot/).
2.6. Reduction of viral cytopathic effect by galectin-3
MDCK cells were infected with influenza A virus (MOI =0.3) and
simultaneously treated with recombinant galectin-3 (gift from Dr. Fu-Tong Liu, Academia Sinica, Taiwan) for two days, cytopathic effect photographed by reverse light microscopy, viral titer in cultured supernatant determined by plaque assay, as depicted in Methods.
2.7. Generation of MDCK cells expressing NS1 protein
NS1 gene from influenza virus A/Taiwan/CMUH01/2007(H1N1) was amplified by RT-PCR with primer pairs: 5’-TCGTGGATCCGATGGACCCAAACACT-3’ and 5’-GACAGCGGCCGCAACTTCTGACCTAAT-3’. Product was cloned into TriEx-4 Neo vector; recombinant vector carrying NS1 gene was sequenced, then
transfected into MDCK by GenePorter reagent. Stably NS1-expressing cells were selected via long-period incubation with 800 µg/ml of G418 culture medium. To confirm protein expression, lysates of NS1-expressing and vector control cells were mixed 1:1 with 2X SDS-PAGE sample buffer without 2-mercaptoethanol and boiled for 10 min. Lysate proteins resolved by SDS-PAGE were transferred to
nitrocellulose, resulting blots blocked with 5% skim milk, reacted with anti-His antibody (Abcam), then washed three times with TBST (Tris-buffered saline, pH 7.5, 0.1% Tween-20). Immune complexes were detected by horseradish peroxidase-154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177
conjugated goat anti-mouse IgG antibodies, followed by enhanced
chemiluminescence detection(Amersham Pharmacia Biotech). Anit-phospho-Ser727 STAT1 mAb examined MDCK signaling and protein changes induced by aloe-emodin.
2.8. Dual-luciferase reporter assay system
Vector control and NS1-expressing cells were transiently co-transfected with an ISRE firefly luciferase cis-reporter and renilla luciferase control reporter (pRluc-C1) in 6-well plates. Transfected cells, maintained in DMEM and 20% FBS after 5 h
incubation with a mixture of plasmid DNA and transfection reagent, were seeded into 24-well plates with DMEM containing 10% FBS. After overnight incubation, cells were treated with 1 and 10 μg/ml aloe-emodin or 1000 U/ml IFN-α for 4 h; enzyme activity of firefly and renilla luciferase in indicated cells was measured by dual Luciferase Reporter Assay System (Promega) and Luminometer TROPIX TR-717 (Applied Biosystems). ISRE driven firefly luciferase was normalized by internal control renilla luciferase in each assay, then ISRE driven luciferase activity indicated ratio of luciferase activity in treated cells over that in untreated cells.
2.9. Quantifying mRNA expression using real time RT-PCR
Total RNA was isolated from vector control and NS1-expressing cells in the presence and absence of 10 μg/ml aloe-emodin and 3000 U/ml IFN-α via a total RNA purification system kit (Invitrogen); cDNA was synthesized from 1000 ng total RNA with oligo dT primer and SuperScript III reverse transcriptase kit (Invitrogen).
Relative mRNA level of indicated gene expression was rated by two-step quantitative RT-PCR with SYBR Green I. Oligonucleotide primer pairs were (1) forward 5’-CAA CCAGCGGTTGACTTTTT-3’ and reverse 5’-ATCCAGGAAGGCA AACTGAA-3’ 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201
for human PKR, (2) forward 5’-GATGTGCTGCCTGCCTTT-3’ and reverse 5’-TTG GGGGTTAGGTTTATAGCTG-3’ for human 2’-OAS, (3) forward
5’-GCCTTATA ACCTGCCTTTGC-3’ and reverse 5’-AACCGACTGTCTTTCTTCCC-3’ for human galectin-3, (4) forward 5’-AACTGCAACCTTTCGAAGCC-5’-AACCGACTGTCTTTCTTCCC-3’ and reverse TGTC GCCTACTACCTGTTGTGC-3’ for human IFNβ, (5) forward GCCATCAGCAA CAACATAAGC-3’ and reverse
CCGAATCAGCAGCGACTC-3’ for human IFNγ, and (6) forward
5’-AGCCACATCGCTCAGACAC-3’ and reverse 5’-GCCCAAACG ACCAAATCC-3’ for human glyceraldehyd-3-phosphate dehydrogenase (GAPDH). Real-time PCR reaction mixture contained 2.5 μl of cDNA (reverse transcription mixture), 200 nM of each primer in SYBR Green I master mix (LightCycler TaqMAn Master, Roche Diagnostics). PCR used amplification protocol consisting of 1 cycle at 50°C for 2 min, 1 cycle at 95°C for 10 min, 45 cycles at 95°C for 15 sec, and 60°C for 1 min. Specific products were amplified and detected in ABI PRISM 7700 sequence
detection system (PE Applied Biosystems), relative changes in mRNA levels of PKR and 2’-5’-OAS normalized by housekeeping gene GAPDH.
2.10 Statistical analysis
Each bar on the graph shows mean of three independent experiments; error bars represent standard error of mean. Data are analyzed by Chi-square and student’s t-test, statistical significance between cell types noted at P < 0.05 (*) or P <0.005 (**).
3. Results
3.1. 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 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226
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) at1 μg/ml concentration; 48 h incubation showed more than 50% rounding of cytopathic effect in infected MDCK versus mock cells (Fig. 2A-a,b). Besides, these anthraquinones markedly reduced cytopathic effect (Fig. 2A-b to e). Virus yield in culture supernatants of infected MDCK was analyzed for relative viral RNA genome level by quantitative real-time RT-PCR (Fig. 2B). Aloe-emodin showed strongest inhibition of virus yield among three anthraquinones (Figs. 2A-c to -e and Fig. 2B). Aloe-emodin treatment caused more than 1-log reduction (equal to 90% effective concentration [EC90]) in virus RNA loads. Subsequent plaque assay
determined half maximal inhibitory concentration (IC50) value of aloe-emodin on
virus yield (Fig. 2B). Aloe-emodin showed dose-dependent inhibition of virus-induced cytopathic effect (Fig. 3A). Infected cells showed about 50% cytopathic effect, those treated with aloe-emodin at concentration of 1 and 2.5μg/ml less than 10% (Fig. 3A). Plaque assay showed 0.5 μg/ml aloe-emodin reducing virus yield, plus inhibition over 60% (Figs. 3B-C). Plaque assay confirmed 1 μg/ml of aloe-emodin reducing virus yield by over 90%: i.e., viral load ascertained by real-time RT-PCR (Figs. 3B-C). Therapeutic index (CC50/IC50) of aloe-emodin pretreatment exceeded 100, suggesting aloe-emodin as antiviral agent against influenza A.
3.2. Up-regulation of galectin-3 by aloe-emodin
To identify key antiviral response, differential MDCK protein expression in the absence or presence of aloe-emodin was tested by 2D gel electrophoresis and LC/ESI-Q-TOF to ferret out differentially regulated proteins. Figure 4 shows protein spots with significant up- or down-regulation excised, digested by trypsin, then 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251
applied into LC/ESI-Q-TOF MS. Up-regulated galectin-3 and thioredoxin, as well as
down-regulated nucleoside diphosphate (NDP) kinase A, were identified with confidence by MASCOT search algorithm (Table 1). MS analysis of galectin-3 indicated Mascot score of 360, sequence coverage of 35%, and four peptide matches. Western blot averred aloe-emodin up-regulating galactin-3 expression in both MDCK and human promonocytes (Figs. 4C-D). PANTHER Classification (Table 1) indicates galectin-3, beta-galactoside-binding animal lectin, involved in cellular processes of immune system, induction of apoptosis, and cell adhesion, portending them as key proteins in aloe-emodin antiviral activity.
3.3. Inhibitory ability of galectin-3 on in vitro replication of influenza A virus
To analyze effect of aloe-emodin on galectin-3 and antiviral gene expression in infected cells, quantitative real-time PCR tested relative mRNA of indicated genes (Fig. 5A). Aloe-emodin induced greater than 2-fold increase of galectin-3, IFNβ, PKR, and IFNγ mRNA in influenza A-infected compared to non-treated infected cells. Recombinant galectin-3 significantly elevated antiviral genes like IFNβ, IFNγ, PKR, and 2',5'-OAS in infected versus non-treated infected cells (Fig. 5B). At 10 μg/ml it reduced cytopathic effect on infected cells from 80% to 10% at 36 h (Fig. 6A), reducing virus yield by over 50%, as determined by plaque assay (Fig. 6B). Data
indicate up-regulation of galectin-3 as crucial to antiviral action of aloe-emodin.
3.4. Signaling induction of aloe-emodin in Influenza A NS1-expressing cells
Since galectin-3 exhibited cytokine-like regulatory action via
JAK/STAT pathways (Jeon et al., 2010), cytokine-like regulatory actions of aloe-emodin were analyzed. Influenza A NS1 manifests Type I IFN antagonistic function (Hayman et al., 2007; Krug et al., 2003; García-Sastre, 2001), so we tested IFN signal response of NS1-expressing cells in the presence or absence of 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276
aloe-emodin. MDCK cells were transfected with plasmid pcDNA3.1-fluA NS1 or empty control vector, followed by two-week treatment with G418 to select stably transfected cells, expression of NS1 detected by immunofluorescent staining and Western blot (data not shown). Subsequent aloe-emodin-induced phosphorylation of STAT1 was rated by Western blot 2 h after treatment. Both types showed Aloe-emodin up-regulating STAT1 phosphorylation (Fig. 7A). ISRE-driven promoter activity of vector control and NS1-expressing cells in response to IFN-α and aloe-emodin were detected by dual-luciferase reporter assay (Fig. 7B), cells
co-transfected with ISRE-driven firefly luciferase reporter plasmid and internal renilla luciferase reporter. After 4 h treatment, relative expression of ISRE-driven firefly luciferase was normalized by renilla luciferase. Firefly luciferase intensity revealed aloe-emodin raising ISRE-driven promoter activity in a dose-dependent manner in both vector control and NS1-expressing cells. Yet IFN-α induced no marked increase of ISRE-driven firefly luciferase NS1-expressing versus mock
controls without treatment. To gauge effect of aloe-emodin on IFN stimulating
gene expression, mRNA levels of PKR and 2’-5’-OAS in NS1-expressing and vector control cells in the presence or absence of aloe-emodin were quantified by real time RT-PCR (Figs. 7C-D). After normalization to GAPDH, aloe-emodin
raised PKR mRNA levels by 49.1-fold in vector control and 34.3-fold in
NS1-expressing cells. IFN-α elicited a 102.6-fold increase of PKR mRNA in vector controls, only slight increase in NS1-expressing cells. Patterns of 2’-5’-OAS mRNA in response to aloe-emodin and IFN-α resembled those of PKR mRNA in vector control and NS1-expressing cells (Fig. 7D), which suggest aloe-emodin having a distinct mechanism from IFN-α to counteract Type I IFN antagonism of Influenza A NS1, linked with antiviral ability of aloe-emodin via galectin-3 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301
against Influenza A.
Discussion
This study documents aloe-emodin inhibiting Influenza A replication and virus-induced CPE (Figs 2-3). 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 g/ml) was more potent than that in direct virucidal assay (Wright and Webster, 2001). Aloe-emodin was observed directly binding with virus envelope: e.g., herpes simplex Types 1-2, varicella-zoster, pseudorabies, influenza (Baritaki and Bonavida, 2010). Its antiviral action shows no type specificity. Proteomic analysis hinted its galectin-3 up-regulation as vital to antiviral mechanism (Fig. 4); potent anti-influenza A activity of recombinant galectin-3 supports the hypothesis (Fig. 6).
Quantitative real time PCR indicated aloe-emodin and galectin-3
up-regulating expression of galectin-3, IFN-γ, IFN-β, PKR and OAS in influenza A virus infected cells (Fig. 5), which portends aloe-emodin up-regulating the galectin-3 expression, linking with induction of IFN-γ and IFN-β as well as IFN-like actions to activate STAT1 phosphorylation and stimulate IFN-inducible gene expression of PKR and 2’-5’-OAS in NS1-expressing or influenza A-infected cells (Fig. 7). Galectin-3 demonstrates IFN-γ-like cytokine, exhibiting IFN-γ receptor 1-dependent activation of JAK-STAT cascade (Park et al., 2011; García-Sastre, 2001). Findings also imply aloe-emodin antiviral mechanism differing from IFNα-induced JAK-STAT signal pathways. Among proteins up-regulated by aloe-emodin, galectin-3, a β-galactoside-binding lectin, has little information concerning antiviral activity. We first observed activity of galectin-3 against influenza A (Fig. 6). Aloe-emodin reversed STAT1-mediated response of NS-1-modulated suppression (Fig. 7), as previously reported:
galectin-3 triggering cytokine-like regulation to activate JAK-STAT cascade in IFN-302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326
γ-deficient cells (Jeon et al., 2010). Galectin-3 is cited as involved in cellular uptake of parvovirus (Garcin et al., 2012). Galectin-1 treatment reduced production of herpes simplex virus-induced proinflammatory cytokines and chemokines (Rajasagi et al., 2012). Chicken galectin-1A and -2 as well as human galectin-1 and -8 promotes
influenza virus binding with cell surface, but not internalization stage (Chernyy et al., 2011). Up-regulation of galectin-1 in lungs of infected mice shows resistance to influenza A: binding to viral surface, then inhibiting virus infectivity (Yang et al., 2011). Besides galectin-3, aloe-emodin up-regulated thioredoxin (Fig. 5 and Table 1). Recombinant thioredoxin-1 attenuates the production of tumor necrosis factor-α and chemokine ligand 1 in lungs of influenza A-inoculated mice, improving their survival (Yashiro et al., 2013). Together with results cited in literature survey, up-regulation of thioredoxin could be responsible for antiviral mechanism of aloe-emodin against influenza A. Anti-influenza A roles of thioredoxin merit further investigation.
Influenza A virus NS1 efficiently suppresses Type-I IFN production, partly by blocking TRIM25 and Riplet ubiquitin E3 ligase activity, inhibiting Lys63-linked ubiquitination of RIG-I (Garcin et al., 2012). NS1 inhibits IFN-induced STAT nuclear translocation and phosphorylation via inhibiting expression of IFN receptor IFNAR1 (Jia et al., 2010), impeding activation of IFN-inducible antiviral2’-5’-OAS and PKR (Krug et al., 2003; García-Sastre, 2001) to down-regulate host innate IFN-mediated antiviralresponse during infection (Hayman et al., 2007). NS1 also activates
phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathways, affecting apoptosis of infected cells (Ehrhardt et al., 2007). Double-strand RNA (dsRNA) up-regulates the galectin-9 expression by activating PI3K and IRF3, but not NF-κB and p38 MAPK (Imaizumi et al., 2007), implying NS1 has no influence on galectin-3 up-regulation induced by aloe-emodin. Hence, aloe-emodin and its analogs spawn antiviral agents 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351
against animal-borne emerging zoonotic influenza, reducing economic loss.
Acknowledgment
We thank the National Science Council (Taiwan) and China Medical University for financial supports (NSC101-2320-B-039-036-MY3 and CMU100-ASIA-16).
Funding: National Science Council of R.O.C. (Taiwan), China Medical University.
Competing interests: None declared. Ethical approved: Not required.
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Figure legends
Fig. 1. Cytotoxicity of aloe-emodin, emodin and crysophanol on MDCK cells, using MTT method. Structures of aloe-emodin, emodin and crysophanol were listed (A), cells treated with various concentrations of aloe-emodin or other tested compounds for 48 h, followed by MTT assay (B), cell survival calculated from ratio of OD570-630 value of treated versus untreated cells. Quadruplicate wells were analyzed for each concentration.
Fig.2. Reduction of virus-induced cytopathic effect and virus yield by aloe-emodin, emodin and crysophanol. MDCK cells infected with Influenza A virus at MOI of 1 in the presence or absence of indicated compound. Cytopathic effect of influenza A virus was photographed 48 h post-infection (A).In the figure list, mock MDCK cells (a), infected cells (b), as well as those infected and treated with aloe-emodin (c), emodin (d) or chrysophanol (e), are shown. Virus yield in cultured medium was collected, RNA genomes extracted, and real-time RT-PCR performed. PCR product levels were monitored with ABI PRISM 7000 sequence detection system (B).
Fig. 3. Dose-dependent inhibition of aloe-emodin on cytopathic effect and virus yield in vitro. MDCK cells were infected with Influenza A virus at a MOI of 1 with various amounts of aloe emodin. After 48-h infection, CPE of influenza A virus-infected and mock cells was observed by inverse microscopy (A), virus yield derived by plaque assay (B). Viral inhibition percentage was analyzed as mentioned in Methods section 2.4. (C).
Fig. 4. Protein profile of MDCK cells in response to aloe-emodin treatment. 100 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498
μg of total protein from (A) mock control or (B) aloe-emodin-treated cells was applied to two-dimensional gel electrophoresis by nonlinear Immobiline DryStrip and 12% polyacrylamide gels. Protein size markers shown at left of each gel were calculated in kDa. Western blot analyzed galectin-3 expression in MDCK (C) and human promonocyte HL-CZ (D) with(out) various amounts aloe-emodin. Lysates were analyzed by 10% SDS-PAGE prior to blot transfer, resulting blot reacted with anti-galectin-3 and anti-β actin antibodies, immunoreactive bands developed with enhanced chemiluminescence substrate.
Fig. 5. Relative mRNA of galectin-3 and antiviral genes in infected cells with(out)
aloe-emodin or galectin-3 treatment. MDCK were infected with influenza A virus at MOI of 0.05 and forthwith treated with aloe-emodin (10 µg/ml) (A) or recombinant galectin-3 (1 µg/ml) (B) for 24 h, then harvested for RNA extraction. Total RNA isolated from each group and real-time RT-PCR was derived as described in Materials and Methods.
Fig. 6. Antiviral activity of galectin-3 in vitro. MDCK cells infected with influenza A virus (MOI = 1) were treated with various amounts of recombinant galectin-3. After 48-h infection, CPE of infected and mock cells was observed by inverse microscopy (A), virus yield calculated by plaque assay (B).
Fig.7. Aloe-emodin-induced activation of STAT1-meidated response in NS1-expressing cells. Stably transfected MDCK with plasmid pcDNA3.1-fluA NS1 or empty control vector were treated with various amounts of aloe emodin. For rating STAT1 phosphorylation (A), lysates from treated cells were analyzed by 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523
10% SDS-PAGE prior to blot, resultant blot probed with anti-phospho-STAT1 (Ser727) and anti-STAT1 antibodies. To test ISRE-driven promoter activity (B), vector control and NS1-expressing cells were transiently co-transfected with internal control reporter pRluc-C1 plus cis-reporting plasmid pISRE-luc, then treated with(out) aloe-emodin and IFN-α for 4-h incubation. Relative firefly luciferase activity was derived by dual Luciferase Reporter Assay System and Luminometer TROPIX TR-717. To detect PKR (C) and 2’-5’-OAS (D) mRNA expression, PKR, 2’-5’-OAS and GAPDH mRNAs were quantifiedwith real-time
PCR, relative mRNA level calculated as ratio of PKR and 2’-5’-OAS mRNA
expression in treated versus untreated cells. 524 525 526 527 528 529 530 531 532 533
