Antiviral Activity of Oroxylin A against Coxsackievirus B3 Alleviates Virus-Induced Acute Pancreatic Damage in Mice
Antiviral Activity of Oroxylin A against Coxsackievirus B3 Alleviates Virus-Induced Acute Pancreatic Damage in Mice
Bo-Eun Kwon 1 2
Jae-Hyoung Song 1 2
Hyuk-Hwan Song 2
Ju Won Kang 0 2
Sam Noh Hwang 0 2
Ki-Jong Rhee 0 2
Aeri Shim 1 2
Eun-Hye Hong 1 2
Yeon-Jeong Kim 2 3
Sang- Min Jeon 2
Sun-Young Chang 2
Dong-Eun Kim 2
Sungchan Cho 2
Hyun-Jeong Ko 1 2
0 Department of Biomedical Laboratory Science, Yonsei University , Wonju, 220-710 , Republic of Korea
1 Laboratory of Microbiology and Immunology, College of Pharmacy, Kangwon National University , Chuncheon, 200-701, South Korea, 2 Agency for Korea National Food Cluster (AnFC), Iksan , Korea
2 Editor: Je-Min Choi, Hanyang University , REPUBLIC OF KOREA
3 College of Pharmacy, Inje University , Gimhae, 621-749, South Korea , 5 College of Pharmacy, Ajou University , Suwon, 443-749, South Korea , 6 Anticancer Agent Research Center, Korea Research Institute of Bioscience & Biotechnology , Cheongju , South Korea
The flavonoids mosloflavone, oroxylin A, and norwogonin, which were purified from Scutellaria baicalensis Georgi, significantly protected Vero cells against Coxsackievirus B3 (CVB3)-induced cell death. To investigate the in vivo antiviral activity of oroxylin A, we intraperitoneally inoculated CVB3 into 4-week-old BALB/c mice. Body weights and blood glucose levels of the mice were decreased after CVB3 infection, and these changes were attenuated by the administration of oroxylin A. Importantly, treatment of mice with oroxylin A reduced viral titers in the pancreas and decreased the serum levels of the inflammatory cytokines including interleukin-6 (IL-6) and tumor necrosis factor (TNF)-α. Additionally, the administration of oroxylin A mitigated the histological pancreatic lesions and apoptotic cell death induced by CVB3 infection and increased the levels of phospho-eIF2α in infected pancreata. The results suggest that oroxylin A may represent a potent antiviral agent against CVB3 infection.
Data Availability Statement; All relevant data are within the paper
Coxsackievirus B (CVB) is associated with more critical diseases, including myocarditis,
pericarditis, meningitis, and pancreatitis, and can cause spastic paralysis. CVB comprises 6
serotypes denoted CVB1–CVB6. CVB3 is an important human pathogen that causes acute and
chronic viral myocarditis in children and young adults and was reported to be associated with
30–50% of all myocarditis cases [
Oroxylin A is an O-methylated flavone which can be found in the medicinal plant
Scutellaria baicalensis, which also contains baicalein, baicalin, wogonin, norwogonin, oroxylin A,
and β-sitosterol [
]. The pharmacological properties of oroxylin A have been widely studied.
design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Oroxylin A has been reported to have multiple functions, including anti-inflammatory,
], and anti-thrombotic activities. Oroxylin A stabilizes p53 through SIRT3-mediated
deacetylation via PTEN dependent manner [
], and also induces apoptosis in human cancer cell line
through translocation of p53 to mitochondria [
]. Moreover, in a report associated with
antiinflammatory effect, oroxyloside, a metabolite of oroxylin A, was shown to have preventive effect
against dextran sulfate sodium-induced colitis via inhibition of NF-κB pathway [
Recently, several studies also have shown broad-spectrum antiviral activities of extracts and
compounds from S. baicalensis. Ethyl acetate and chloroform extracts of S. baicalensis were
reported to show inhibitory activity against the neuraminidase enzyme of influenza virus, and
this extract, as well as one of its major components, baicalein, demonstrated in vitro antiviral
activity against influenza viruses including the pandemic 2009 H1N1 and seasonal 2007 H1N1
]. S. baicalensis also showed moderate or weak antiviral activity against respiratory
syncytial virus (RSV), largely through wogonin and oroxylin A [
]. However, although all of
these reports showed that bioactive flavones including wogonin and baicalin have antiviral
activities, few studies have suggested an antiviral activity of oroxylin A [
In the current study, we assessed whether extracts of S. baicalensis have antiviral activity
against CVB3 infection, and we found that oroxylin A and the chloroform fraction of S.
baicalensis, which principally contains oroxylin A showed significant antiviral activities against
CVB3 infection in vitro and in vivo. In addition, we found that the inflammatory mediators
interleukin (IL)-6, chemokine (C-C motif) ligand 2 (CCL2), chemokine (C-X-C motif) ligand 1
(CXCL1), and tumor necrosis factor (TNF)-α were increased in serum after CVB3 infection in
mice, but administration of oroxylin A to infected mice significantly reduced the elevation of
these cytokines and chemokines. Oroxylin A showed antiviral activity associated with a
reduced level of CVB3-induced cytotoxicity, which might be mediated by an increase in eIF2α
phosphorylation in response to endoplasmic reticulum (ER) stress. Collectively, our results
indicated that oroxylin A has the potential to be an antiviral agent against CVB3 infection.
Materials and Methods
Isolation of active compounds from the aerial parts of Scutellaria
The dried aerial parts of Scutellaria baicalensis Georgi (1.2 Kg) were cut into pieces and
extracted with methanol (3 × 2 L) in an ultrasonic apparatus at room temperature after
evaporation of the solvent. The methanol extract was suspended in H2O and successively partitioned
into chloroform, ethyl acetate, n-butanol, and water fractions after removal of the solvents
under vacuum. Then, these fractions were subjected to a sulforhodamine B (SRB)-based
antiviral activity assay (Table 1), and we found that the chloroform fraction had antiviral activity
against CVB3. Next, the chloroform fraction was subjected to C18 column silica gel column
adsorption chromatography (40–63 μm, 300 g) (Merck & Co., Kenilworth, NJ, USA), and
eluted with a gradient consisting of methanol:water (3:7, 4:6, 6:4, 8:2, and 10:0; 2 × 500 ml).
The fraction was separated on a Sephadex LH-20 column (Sigma-Aldrich, St. Louis, MO, USA)
using 100% methanol, and norwogonin, oroxylin A, and mosloflavone were obtained. The
structures of norwogonin, oroxylin A, and mosloflavone were identified using electrospray
ionization mass spectrometry, 1H-nuclear magnetic resonance (NMR), and 13C-NMR.
Cell lines and viruses
CVB3 virus (ATCC VR-30) was obtained from the Division of Vaccine Research of the Korea
Centers for Disease Control and Prevention (KCDC, Cheongwon, Korea), and was propagated
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at 37°C in Vero cells (ATCC, Manassas, VA, USA), a kidney epithelial cell line that originated
from an African green monkey. Vero cells were maintained in minimal essential medium
supplemented with 10% (v/v) fetal bovine serum and 1% (v/v) antibiotic-antimycotic solution.
Gibco1 brand antibiotic-antimycotic solution, trypsin-ethylenediaminetetraacetic acid, fetal
bovine serum, and minimal essential medium were purchased from Life Technologies,
Carlsbad, CA, USA, and Falcon™ tissue culture plates were purchased from BD Biosciences, San
Jose, CA, USA.
Wild-type inbred BALB/c mice were purchased from Charles River Laboratories (via Orient
Bio Inc., Sungnam, Korea). Mice were maintained under specific pathogen-free conditions in
the experimental facilities at Kangwon National University. The experiments were approved by
the Institutional Animal Care and Use committees of Kangwon National University (Permit
Number: KW-140811-2). Female 4-week-old BALB/c mice (n = 4/group) were
intraperitoneally inoculated with 1 × 106 TCID50, the half-maximal 50% tissue culture infective dose of
CVB3 in 200 μl. Groups of mice were checked for the body weight change every morning until
the end of experiments. The mice were anesthesed with a mixture solution of 9:1
xelazine/ketamine and sacrificed by CO2 inhalation followed by cervical dislocation. BALB/C mice infected
with CVB3 were administered intraperitoneally oroxylin A (10 mg/kg) for 5 days.
The antiviral activities and cytotoxicities of the test extracts and compounds were evaluated by
using the SRB method to measure the cytopathic effect (CPE) induced by viral infection as
previously reported [
]. The Time-of-addition (TOA) assay was designed to determine the
mechanism of action of antiviral compounds [
]. The active compounds were then added onto
the cells at 30 μg/ml either before (-1 h), during (0 h), or after (1, 2, 4, and 8 h) periods of
CVB3 infection. After 48 h, SRB assay was performed.
Plasmid p53CB3-LUC [
] which contains the firefly luciferase gene in place of the P1
capsidcoding region of the CVB3 viral genome, was kindly provided by Frank J. M. van Kuppeveld
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(Utrecht University, Netherlands). Plasmid p53CB3-LUC was linearized by Mlu I enzyme and
used for the production of CVB3 replicon RNAs by using the Ribomax large-scale RNA
production system (Promega). To examine the effect of oroxylin A on the replication of CVB3
replicon, 293T cells (3.5 × 105 cells/well) in a 6-well plate were transfected with 0.4 μg CVB3
replicon RNA using Lipofectamine 2000 (Invitrogen), split into 96-well plates (2 × 104 cells/
well), and simultaneously treated with 10 μg/ml oroxylin A or 10 μM rupintrivir (as a positive
control). Eight hours after treatment, the cells were assessed for firefly luciferase activity using
the One-Glo Luciferase assay kit (Promega). In the same conditions, another set of CVB3
replicon-transfected cells was assayed for cell viability using CellTiter-Glo Luminescent cell viability
assay kit (Promega) [
Cell viability assay
Vero cells were seeded in 96-well plates at a density of 3 x 104 cells per well and incubated for
24 h. Vero cells were infected with CVB3 and treated with salubrinal at the indicated
concentrations. After incubation for indicated time, cell viability was measured by SRB assay [
Histology and TUNEL assay
The pancreata of mice were removed and washed with PBS before being fixed with 4% (w/v)
formaldehyde for overnight. The tissues were embedded in paraffin, cut into 5 μm sections,
and stained with hematoxylin and eosin (H&E). The 5 μm paraffin-embedded section were
stained using an Apoptag1 peroxidase in situ detection kit (catalogue number S7100,
Chemicon, Billerica, MA, USA) to determine the level of apoptosis by TUNEL assay according to the
manufacturer’s instructions. The number of TUNEL-positive cells was counted by a
pathologist using a blind test.
Cytokine measurement (ELISA)
The levels of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α) and chemokine (C-C
motif) ligand 2 (CCL2) were determined using a mouse ELISA Ready-SET-GO kit
(ebioscience). The levels of chemokine (C-X-C motif) ligand 1 (CXCL1)/KC measured using a
DuoSet1 mouse ELISA kit (R&D Systems, Minneapolis, MN, USA) and an ELISA MAX™
standard kit (Biolegend, Inc., San Diego, CA, USA), respectively. Experiments were performed
according to each manufacturer’s instructions.
Rabbit anti-cytoskeletal actin (Bethyl Laboratories, Montgomery, TX, USA), Mouse anti-CVB3
VP1 (Dako, Copenhagen, Denmark), Rabbit anti-eIF2α (Cell signaling Technology, Inc.),
Rabbit anti-phospho-eIF2α (Ser51) (Cell signaling Technology, Inc.) and Rabbit anti-ATF4
(CREB-2) (Santa Cruz Biotechnology, Inc.) were used. The enhanced chemi-luminescence
substrate femtoLUCENT™ PLUS-HRP (G-Biosciences, St. Louis, MO, USA) was applied and
images of bands were captured using an ImageQuant™ LAS 4000 Mini system (GE Healthcare
Life Sciences, Little Chalfont, Buckinghamshire, UK). Quantification of band densities was
performed using Image J software (NIH, Bethesda, MD, USA).
Real-time polymerase chain reaction (PCR)
Total RNA was extracted from Vero cells and pancreata of mice using a QIAamp1 viral RNA
mini kit (Qiagen, Limburg, Holland). Reverse transcription was performed using SuperScript™
II reverse transcriptase (Invitrogen, Grand Island, NY, USA) according to the manufacturer’s
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instructions. For real-time PCR analysis, the cDNA was serially diluted 10-fold and amplified
using a 7500 real time PCR system (Applied Biosystems, Foster City, CA) with Power SYBR1
Green PCR master mix (Applied Biosystems). We used the following primers: β-actin (sense
50-CCA TCA TGA AGT GTG ACG TGG-30, antisense 50-GTC CGC CTA GAA GCA TTT
GCG-30) and EV-NCR (sense 50-CCG GCC CCT GAA TGC GG-30, antisense 50-ATT CTT
TAA TTG TCA CCA TAA GCA GCC A-30).
To compare multiple groups, we carried out one-way ANOVA followed by the Tukey post hoc
test using GraphPad Prism version 5 software (Graphpad, San Diego, CA, USA). Values of
p < 0.05 were considered significant at a 95% confidence interval.
In vitro antiviral activity of S. baicalensis against CVB3 infection
We found that the extract of S. baicalensis significantly protected against decreases in cell
viability caused by CVB3 infection (Table 1). The methanol extract of S. baicalensis (IC50 =
9.53 μg/ml) potently inhibited CPE caused by CVB3 infection. To identify the active antiviral
compounds, the methanol extract was further fractionated into chloroform, ethyl acetate,
butanol, and water fractions, and we found that antiviral activity was highly retained in the
chloroform fraction. The IC50 value of the chloroform fraction in CVB3-infected Vero cells was
determined to be 9.76 μg/ml. Thus we undertook further studies to identify the antiviral
compounds present in the methanol extract of S. baicalensis.
In vitro antiviral activities of norwogonin, oroxylin A, and mosloflavone
against CVB3 infection
After further purification, we obtained several compounds with potential antiviral activities
against CVB3, including norwogonin, oroxylin A, and mosloflavone. The in vitro antiviral
activities of these compounds were assessed (Fig 1A). All 3 compounds showed significant
antiviral activity against CVB3 infection at concentrations 10 μg/ml. Although mosloflavone
exhibited approximately 20% of cytotoxicity at 50 μg/ml, oroxylin A and norwogonin showed
no detectable cytotoxicity against Vero cells at concentrations < 50 μg/ml, which is
contradictory to several recent study showing that oroxylin A is able to induce apoptosis. However, most
of them used higher concentration of oroxylin A (> 100 μg/ml) than we used, and there could
be cell-type dependency [
The CC50, IC50, and therapeutic index (TI) values of norwogonin, oroxylin A, and
mosloflavone are shown in Table 2. We analyzed the CPE inhibiting activities of norwogonin, oroxylin
A, and mosloflavone on Vero cells infected with CVB3. In the absence of CVB3 infection, Vero
cells treated with vehicle (Fig 1B-a) or 50 μg/ml of each compound including norwogonin (Fig
1B-b), oroxylin A (Fig 1B-c), and mosloflavone (Fig 1B-d) showed typical spread-out shapes
with normal morphology. Infection with CVB3 in the absence of drug treatment resulted in a
severe CPE (Fig 1B-e). Addition of norwogonin (Fig 1B-f), oroxylin A (Fig 1B-g), and
mosloflavone (Fig 1B-h) to the Vero cells infected with CVB3 prevented any visible CPE from
occurring. These results suggested that norwogonin, oroxylin A, and mosloflavone from S.
baicalensis had significant antiviral activity against CVB3 without inducing cytotoxicity in
To determine whether the increase in cell viability upon norwogonin, oroxylin A, and
mosloflavone treatment in CVB3-infected cells was due to direct antiviral activity of the drug, we
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Fig 1. The antiviral activities of norwogonin, oroxylin A, and mosloflavone against CVB3 in vitro. (A)
Antiviral activities of norwogonin, oroxylin A, and mosloflavone against CVB3 in Vero cells were measured by
SRB assay. The indicated concentration of norwogonin, oroxylin A, and mosloflavone, ranging from 0.4–
50 μg/ml, were added to Vero cells infected with the CCID50 titer of CVB3. Cells were cultured for 48 h and
the antiviral activity was determined by CPE reduction assay. Absorbance values are presented as
means ± SD from 3 independent experiments each carried out in triplicate. **P<0.001; ***P<0.0001 using
one-way ANOVA with Tukey’s post hoc test. (B) The effects of norwogonin, oroxylin A, and mosloflavone on
CVB3–induced CPE using SRB assay. The virus-infected cells were treated with norwogonin, oroxylin A, and
mosloflavone at 50 μg/mL. After incubation at 37°C in 5% CO2 for 48 h, the morphologies of cells were
photographed under a microscope. (B-a) Non-infected cells; (B-b) non-infected cells treated with norwogonin;
(B-c) non-infected cells treated with oroxylin A; (B-d) non-infected cells treated with mosloflavone; (B-e)
CVB3-infected cells; (B-f) CVB3-infected cells treated with norwogonin; (B-g) CVB3-infected cells treated
with oroxylin A; (B-h) CVB3-infected cells treated with mosloflavone; (C) Relative CVB3 gene expression in
control, CVB3-infected, and 10 μg/ml norwogonin-, oroxylin A-, and mosloflavone-treated cells by real-time
PCR. **P<0.001 using one-way ANOVA with Tukey’s post hoc test. (D) TOA effects of norwogonin, oroxylin
A, and mosloflavone on CVB3 replication in Vero cells. 30 μg/mL of each compound was added either during
(0 h), or after (1, 2, 4, or 8 h) virus infection. After 2 days, inhibition was evaluated by the SRB method and
expressed as the inhibition rate. Percentage values represent the mean ± SD of 3 independent experiments.
b Concentration required to inhibit virus-induced CPE by 50% (μg/mL).
c Therapeutic index = CC50/IC50
assessed CVB3 5' non-coding region (NCR) mRNA level 48 h after infection. The expression of
5' NCR transcript was decreased by norwogonin, oroxylin A, and mosloflavone (10 μg/ml) as
compared to vehicle treatment (Fig 1C), suggesting that CVB3 replication was inhibited by
norwogonin, oroxylin A, and mosloflavone.
To further characterize the antiviral activities of these compounds, we assessed their
inhibitory effects on the CPE caused by CVB3 infection of Vero cells when the compounds were
added at different times before, with, and after CVB3 inoculation (Fig 1D). This TOA assay is
intended to determine the effects of adding the test compounds at different stages during the
viral replication cycle [
]. The results showed that norwogonin and oroxylin A suppressed
CVB3 infection when they were added just after virus inoculation (0 h) and at the early stages
after virus inoculation (1, 2, 4, and 8 h), whereas mosloflavone only showed antiviral activity
when added within 2 h of CVB3 inoculation. When added at 1 h before CVB3 infection and
washed out before viral infection, norwogonin, oroxylin A, and mosloflavone did not show
antiviral activity. Collectively, we can presume that oroxylin A and norwogonin were effective
at the early stages of viral infection and seems effective even treated 8 h after CVB3 infection,
suggesting that they might be therapeutically used for CVB3 infection.
Oroxylin A inhibits CVB3 proliferation
In order to know whether oroxylin A inhibits the replication of CVB3 in infected cells, Vero
cells were transfected with in vitro-transcribed CVB3-replicon RNAs, simultaneously treated
with a 10 μg/ml of concentration of oroxylin A for 8 h, and then assayed for luciferase activity.
Oroxylin A inhibited the replication of CVB3 replicons at a 10 μg/ml of concentration (Fig
2A). Additionally, it did not exhibited any cytotoxicity on Vero cell in 10 μg/ml concentration,
which was assessed using CellTiter-Glo reagent (Fig 2B). In the same conditions, rupintrivir, a
potent 3C inhibitor, which was used as positive control, showed strong antiviral activity
without cytotoxic effect at 10 μM concentration.
In vivo antiviral activity of oroxylin A against CVB3 infection
To ascertain the antiviral activity of oroxylin A in vivo, BALB/c mice were intraperitoneally
injected with 1 × 106 times the TCID50 titer of CVB3, resulting in pancreatic infection. We
assessed the body weights (Fig 3A) and blood glucose levels (Fig 3B) of CVB3-infected mice.
Fig 2. Oroxylin A inhibits the replication of the CVB3 replicon. (A) Vero cells were transfected with in
vitro-transcribed CVB3-replicon RNAs, immediately treated with the indicated concentrations of oroxylin A for
8h, and then assayed for firefly luciferase activity. The luciferase activity of DMSO-treated cells was
considered to be 100%. **P<0.001 using one-way ANOVA with Tukey’s post hoc test. (B) In the same
conditions, another set of CVB3 replicon-transfected cells was assayed for cell viability using CellTiter-Glo
reagent. The activity of DMSO-treated cells was considered to be 100%.
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Fig 3. The antiviral activity of oroxylin A against CVB3 in vivo. BALB/c mice were infected with a 1 × 106
TCID50 dose of CVB3 and given oroxylin A or vehicle. Body weights (A) and glucose levels (B) were
measured for 5 days. *p < 0.05, **p < 0.01, ***p < 0.001 for CVB3 + oroxylin A versus CVB3 + vehicle, using
one-way ANOVA. Virus titers of mice were determined 5 days post infection by real-time PCR (C) and
western blots (D) in the pancreata. In western blot, densitometric measurement of VP1 expression is
normalized to β-actin.
Body weights and serum glucose levels were decreased after CVB3 infection due to pancreatic
damage, and these infection-induced changes were significantly attenuated by the treatment of
infected mice with oroxylin A. In addition, we measured viral titers in pancreata from infected
mice. Administrations of oroxylin A significantly decreased the level of CVB3 VP1 gene
expression and CVB3 VP1 protein expression compared with that of infected mice
administered with vehicle only (Fig 3C and 3D). These result indicated that oroxylin A exerted antiviral
activity against CVB3-induced pancreatic infection in vivo.
Serum cytokine and chemokine levels in mice treated with oroxylin A
Elevated levels of serum cytokines and chemokines including IL-6, CCL2, and CXCL1 have
been reported to be pathological hallmarks of pancreatitis after CVB3 infection [
analyze the inflammatory status of CVB3-infected mice, serum levels of IL-6, CCL2, CXCL1, and
TNF-α were detected. There were minimal levels of serum IL-6, CCL2, CXCL1, and TNF-α in
mice without CVB3 infection, while those infected with CVB3 had significantly increased
inflammatory cytokines and chemokines on day 5 post infection (Fig 4), which might be
associated with exacerbation of pancreatic inflammation. The treatment of CVB3-infected mice
with oroxylin A decreased the serum levels of those cytokines and chemokines, suggesting
anti-inflammatory effects of oroxylin A upon CVB3 infection.
Oroxylin A prevents damage to the pancreas
For pathological analysis, histology sections were prepared from pancreata of mice. Uninfected
pancreata were histologically normal, but those harvested 5 days after CVB3 infection showed
almost complete ablation of acinar cells, as well as infiltration of inflammatory cells (Fig 5A)
consistent with the decreased level of serum glucose on day 5 post infection. However,
CVB3-infected mice administered with oroxylin A showed reduced histopathological
abnormalities, although they still had some acinar cell hypochromicity and inflammatory cell
infiltration. In addition, we confirmed that the mice administered with oroxylin A were
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Fig 4. Serum cytokine and chemokine levels in mice treated with oroxylin A. Sera were collected on day
5 post infection and levels of IL-6 (A), CXCL1 (B), CCL2 (C), and TNF-α (D) were determined.
*P<0.01;**P<0.001;***P<0.0001 using one-way ANOVA with Tukey’s post hoc test.
significantly protected against the reduction in the numbers of acinar cells that occurred in the
CVB3-infected, vehicle-treated mice (Fig 5B). Using TUNEL assay, we found that the
pancreata of infected mice exhibited apoptotic cell death (Fig 5C and 5D). Oroxylin A treatment of
CVB3-infected mice considerably decreased pancreatic apoptotic cell death and the apoptotic
cells observed in those mice were found outside the pancreatic tissue rather than within it.
These results supported a protective role of oroxylin A against CVB3-induced pancreatic
Fig 5. Administration of oroxylin A prevents damage to the pancreas. (A) Representative H&E staining
of pancreas sections from uninfected mice (a), mice infected with CVB3 (b), and CVB3-infected mice treated
with oroxylin A (c). (Scale bar = 20 μm) (B) Numbers of acini were counted from H&E images of pancreas
sections. ***p < 0.001 as compared with the non-infected (nil) group, and #p < 0.05 for CVB3 + oroxylin A
versus CVB3 + vehicle (one-way ANOVA). (C) Representative image of TUNEL staining (Green
fluorescence) showing the apoptotic cells in the pancreas with red fluorescence from propidium
iodidestained nuclei. Some apoptotic cells were marked with white arrow. (D) TUNEL+ cells were counted in
pancreatic sections. ***p < 0.001 (one-way ANOVA).
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Fig 6. Oroxylin A enhances eIF2α phosphorylation and NF-κB signaling. Vero cells were infected with
CVB3 and treated with oroxylin A (25 μg/ml) for 16 h. (A) Protein levels of phosphorylated eIF2α,
unphosphorylated eIF2α, ATF4, and β-actin as measured by western blots. (B) The virus-infected cells were
treated with salubrinal at the indicated concentrations. After incubation at 37°C in 5% CO2 for 48 h, cell
viability was evaluated by SRB assay. *p < 0.05, **p < 0.01 (one-way ANOVA followed by the Tukey post hoc
Oroxylin A increased phosphorylation of eIF2α
Recently, it was reported that oroxylin A activated the ER stress response through the
doublestranded RNA-dependent kinase (PKR)-like ER kinase (PERK)/eukaryotic translation initiation
factor 2α (eIF2α)/activating transcription factor 4 (ATF4) pathway [
]. We confirmed that
treatment with oroxylin A increased eIF2α phosphorylation in CVB3-infected Vero cells (Fig
6A). To ascertain the role of phosphorylated eIF2α, we assessed the effect of salubrinal, which
was reported to be a blocking agent of eIF2α dephosphorylation [
], and we found that it
inhibited the CVB3-induced CPE in Vero cells (Fig 6B). We confirmed that the treatment of cells with
salubrinal moderately attenuated cytotoxicity induced by CVB3 infection. Collectively, these
results showed that oroxylin A up-regulated eIF2α phosphorylation and attenuated
CVB3-induced cell death and partially reduced acute pancreatic damage by CVB3 infection.
The pancreas is one of the target organs of CVB3 infection together with the liver, heart, brain,
and meninges [
]. Although most CVB3 infections in humans lack significant symptoms,
some CVB3 infections were found to be associated with the development of type 1 diabetes
with chronic inflammatory diseases of the pancreas , which renders CVB3 infection of the
pancreas a clinically significant event [
]. Acinar and beta cells are natural primary targets of
CVB3 infection [
]. Infection with CVB3 induced autophagy-like vesicle formation in
pancreatic acinar cells in mice , in which microtubule-associated protein light chain 3
conversion and large autophagic vesicles called “megaphagosomes” were observed [
]. In the
current study, we confirmed that CVB3 infection in mice resulted in acute pancreatic
inflammation accompanied by an increased viral load in the pancreas and increased levels of
proinflammatory cytokines in serum, and these pathologic changes were significantly
ameliorated by the administration of oroxylin A isolated from the medicinal plant S. baicalensis.
It was reported that some serum cytokines and chemokines were elevated during CVB3
], especially IL-6, CXCL1, and CCL2. In the current study, we measured serum
cytokines and chemokines 5 days post CVB3 infection, and found significant increases in the
levels of IL-6, CXCL1, CCL2, TNF-α. However, we did not detect significant changes in the
levels of other cytokines including IL-4 and IL-10 (data not shown). On the basis of the elevation
of inflammatory cytokines in the serum after CVB3 infection in mice, we decided to evaluate
the levels of those cytokines to further assess the antiviral activity of oroxylin A against CVB3
infection in vivo. We hypothesized that the increased levels of CXCL1 and IL-6 could be driven
by increased nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) signaling as a
result of the increased ER stress caused by CVB3 infection.
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Since CVB3 is a nonenveloped RNA virus that replicates in double-membrane vesicles, we
can predict that CVB3 infection may influence ER-associated cellular processes. Likewise,
many kinds of viruses have been shown to trigger ER stress after infection by utilizing different
molecular pathways associated with the unfolded protein response (UPR) [
]. The UPR
mainly involves 3 interconnected pathways centered on serine/threonine-protein kinase/
endoribonuclease (IRE1)/x-box binding protein 1 (XBP1), PERK/eIF2α, and ATF6,
respectively. CVB3 infection induced apoptosis in HeLa cells, and this was correlated with the
induction of C/EBP homologous protein (CHOP), sterol regulatory element binding protein 1, and
caspase-12. CVB3 infection activated the PERK/eIF2α and IRE1/XBP1 pathways while
suppressing p58IPK, a negative regulator of PKR and PERK [
], in an ATF6a dependent manner
]. Since ER stress responses were previously known to be closely linked with autophagy
pathways, CVB3 might regulate the ER-autophagy pathway to prevent its autophagic
EIF2α can be phosphorylated under several circumstances including starvation, viral
infection, and ER stress. Accordingly, several kinases including GCN2, PKR, and PERK could be
responsible for the phosphorylation of eIF2α. On viral infection, phosphorylation of eIF2α
could occur by the activation of PKR, a dsRNA-dependent protein kinase, which was initially
known as an actor in antiviral response of interferons. Phosphorylation of eIF2α by PKR
results in attenuation of CAP-dependent translation and induces autophagy activation, but not
always ends up with cell death [
]. Interestingly, oroxylin A was reported to activate UPR in
HepG2 cancer cells by the PERK/eIF2α/ATF4 pathway, but excessive activation of this
pathway resulted in the activation of CHOP and induced cell death [
]. In addition, recent studies
have suggested that CVB3 infection induces ER stress, which is generally caused by the
accumulation of misfolded/unfolded proteins in the ER, and that CVB3 residency in ER might be
closely associated with viral replication and pathogenesis. It is known that salubrinal blocks
eIF2α dephosphorylation and inhibits viral replication. It was previously reported that
salubrinal has antiviral activity against herpes simplex virus [
]. With a number of recent studies
having reported associations between eIF2α phosphorylation and antiviral activity, it is
possible that salubrinal or similar compounds may have antiviral effects via regulating the ER stress
response during viral infection [
In conclusion, the results show that administration of oroxylin A exhibited an antiviral
effect against CVB3 infection and consequently decreased serum levels of inflammatory
cytokines and the severity of histological lesions in mice infected with CVB3. We confirmed that
oroxylin A affected the phosphorylation of eIF2α and cell death. Based on these results, we
suggest that oroxylin A could be a candidate for therapeutic use as an antiviral against CVB3
Conceived and designed the experiments: HJK. Performed the experiments: BEK JHS JWK
SNH DEK. Analyzed the data: AS EHH SYC KJR. Contributed reagents/materials/analysis
tools: HHS SMJ. Wrote the paper: SC YJK HJK.
11 / 13
12 / 13
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