Epigoitrin, an Alkaloid From Isatis indigotica, Reduces H1N1 Infection in Stress-Induced Susceptible Model in vivo and in vitro

Abstract

Stress has been proven to modulate an individual’s immune system through the release of pituitary and adrenal hormones such as the catecholamines, growth hormone, and glucocorticoids. These signal molecules can significantly alter the host immune system and make it susceptible to viral infection. In this study, we investigate whether epigoitrin, a natural alkaloid from Isatis indigotica, provides protection against influenza infection by reducing the host’s susceptibility to influenza virus under stress and its underlying mechanism. To support it, the mouse restraint stress model and the corticosterone-induced stress model were employed. Our results demonstrated that epigoitrin significantly decreased the susceptibility of restraint mice to influenza virus, evidenced by lowered mortality, attenuated inflammation, and decreased viral replications in lungs. Further results revealed that epigoitrin reduced the protein expression of mitofusin-2 (MFN2), which elevated mitochondria antiviral signaling (MAVS) protein expression and subsequently increased the production of IFN-β and interferon inducible transmembrane 3 (IFITM3), thereby helping to fight viral infections. In conclusion, our study indicated that epigoitrin could reduce the susceptibility to influenza virus via mitochondrial antiviral signaling.

Full text

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ORIGINAL RESEARCH
published: 07 February 2019
doi: 10.3389/fphar.2019.00078
Edited by:
Ioanna Andreadou,
National and Kapodistrian University
of Athens, Greece
Reviewed by:
Alan G. Goodman,
Washington State University,
United States
Ivan Tattoli,
Columbia University, United States
*Correspondence:
Yi-Fang Li
liyifang706@jnu.edu.cn
Rong-Rong He
rongronghe@jnu.edu.cn
Specialty section:
This article was submitted to
Experimental Pharmacology
and Drug Discovery,
a section of the journal
Frontiers in Pharmacology
Received: 01 October 2018
Accepted: 21 January 2019
Published: 07 February 2019
Citation:
Luo Z, Liu L-F, Wang X-H, Li W,
Jie C, Chen H, Wei F-Q, Lu D-H,
Yan C-Y, Liu B, Kurihara H, Li Y-F and
He R-R (2019) Epigoitrin, an Alkaloid
From Isatis indigotica, Reduces H1N1
Infection in Stress-Induced
Susceptible Model in vivo and in vitro.
Front. Pharmacol. 10:78.
doi: 10.3389/fphar.2019.00078
Epigoitrin, an Alkaloid From Isatis
indigotica, Reduces H1N1 Infection
in Stress-Induced Susceptible Model
in vivo and in vitro
Zhuo Luo1,2 , Li-Fang Liu1,2 , Xiao-Hua Wang1,2, Wen Li1,2, Chong Jie1,2, Huan Chen1,2,
Fan-Qin Wei3, Dan-Hua Lu1,2 , Chang-Yu Yan1,2, Bo Liu4, Hiroshi Kurihara1,2 , Yi-Fang Li1,2*
and Rong-Rong He1,2*
1Guangdong Engineering Research Center of Chinese Medicine & Disease Susceptibility, Jinan University, Guangzhou,
China, 2Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, College
of Pharmacy, Jinan University, Guangzhou, China, 3Department of Otorhinolaryngology, Head and Neck Surgery, The First
Affiliated Hospital, Sun Yat-sen University, Guangzhou, China, 4State Key Laboratory of Biotherapy, West China Hospital,
Sichuan University, Chengdu, China
Stress has been proven to modulate an individual’s immune system through the release
of pituitary and adrenal hormones such as the catecholamines, growth hormone, and
glucocorticoids. These signal molecules can significantly alter the host immune system
and make it susceptible to viral infection. In this study, we investigate whether epigoitrin,
a natural alkaloid from Isatis indigotica, provides protection against influenza infection
by reducing the host’s susceptibility to influenza virus under stress and its underlying
mechanism. To support it, the mouse restraint stress model and the corticosterone-
induced stress model were employed. Our results demonstrated that epigoitrin
significantly decreased the susceptibility of restraint mice to influenza virus, evidenced
by lowered mortality, attenuated inflammation, and decreased viral replications in lungs.
Further results revealed that epigoitrin reduced the protein expression of mitofusin-2
(MFN2), which elevated mitochondria antiviral signaling (MAVS) protein expression and
subsequently increased the production of IFN-βand interferon inducible transmembrane
3 (IFITM3), thereby helping to fight viral infections. In conclusion, our study indicated
that epigoitrin could reduce the susceptibility to influenza virus via mitochondrial antiviral
signaling.
Keywords: epigoitrin, influenza virus, stress-induced susceptibility, MFN2, MAVS
INTRODUCTION
Influenza A viruses (IAVs), highly contagious pathogens, are responsible for severe respiratory
infection in humans and animals worldwide with pandemic potential (Chen et al., 2018). At present,
antiviral drugs and vaccines are the main treatment for IAVs infection. Due to the high mutation
rate and antiviral-drug-resistant strains of IAVs (Kumar et al., 2018), developing vaccines and anti-
viral drugs for IAVs infection are still full of challenges. Hence, there is an urgent need to identify
novel antiviral therapies or complementary strategies.
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Luo et al. Epigoitrin, Stress-Induced Viral Susceptibility
Many herbal extracts or natural products have been
demonstrated to possess potent anti-influenza, preventive
and immunomodulatory effects. The dry root of Isatis indigotica
(Ban Lan Gen, BLG), a traditional Chinese medicine, has been
used for anti-influenza in clinics over thousands of years in
China (Zhou and Zhang, 2013). Chemical studies showed that
BLG contains various compounds such as alkaloids, nucleosides,
amino acids, organic acids (Xiao et al., 2014). Epigoitrin as an
alkaloid was used as a marker compound of BLG in the 2015
edition of the Chinese Pharmacopoeia (National Pharmacopoeia
Commission, 2015). It has previously been reported that
epigoitrin exerts antiviral activity against influenza A1 virus
FM1 via inhibiting virus attachment and multiplication in vitro
(Xiao et al., 2016). However, no in vivo pharmacological studies
confirmed the anti-influenza activities. Our previous studies
indicated that restraint stress could increase the susceptibility
to the influenza virus in mice and provide a useful model
basis for evaluating the effectiveness of the herbal medicinal
product and natural products (He et al., 2011;Tang et al., 2014;
Chen et al., 2017). It is well known that stressful events take
a toll in the development of disease, especially in infectious
disease. Stressors can increase susceptibility to infectious agents,
dysregulate the humoral and cellular immune responses to
pathogens and increase the risk of catching infectious diseases.
Restraint is a commonly used stressor for mice. Mice are placed
in tubes with holes such that they can breathe and move forward
or backward but cannot turn around, which is often applied
overnight during the most active time for mice (Glaser and
Kiecolt-Glaser, 2005). Moreover, influenza and pneumonia are
the fifth leading cause of death among individuals over 50 years
old, which was related to greater immunological impairments
associated with distress or depression in the old than that in
the young (Glaser and Kiecolt-Glaser, 2005). Accordingly,
stress-related immune disorders may be a core mechanism
behind multiple infectious diseases, and if antiviral drugs
or compounds have the ability to regulate stress-mediated
immune disorders, they might play a more important role in
the treatment of influenza. In this study, we employed the
restraint-stress induced susceptible model to investigate the
preventive effects of epigoitrin on influenza infection and its
related mechanisms.
MATERIALS AND METHODS
Compounds
Epigoitrin with 98% purity was purchased from Aladdin
Biochemical Technology Co., Ltd. (Shanghai, China).
Oseltamivir was obtained from Yichang Changjiang
Pharmaceutical Co., Ltd. (Wuhan, China). Corticosterone
was purchased from Sigma (MO, United States).
Virus
The human HlN1 prototype strain, mouse-adapted A/FM/1/47
virus (Smeenk and Brown, 1994), was provided by College of
Veterinary Medicine of South China Agricultural University
(Guangzhou, China). Viruses were propagated in the allantoic
cavities of specific-pathogen-free fertilized eggs. The allantoic
fluid containing virus was harvested and stored in aliquots at
−80◦C until used. Median tissue culture infective dose (TCID50)
was measured in MDCK cells and calculated according to the
Reed-Muench formula after serial dilution of the stock. Amounts
of 10 TCID50 value were used for viral infection in all the cell
experiments.
Mice and Experimental Design
Specific-pathogen-free male Kunming mice with 4 weeks of age
and weighing 12–15 g were purchased from Guangdong Medical
Laboratory Animal Center (Guangzhou, China). The animals
performed in this study were housed in plastic cages and lived
under standard laboratory conditions. Animal experiments were
approved by the Animal Care and Use Committee of Jinan
University (Approval ID: SYXK 20150310001) and performed in
compliance with the National Institute of Health’s Guide for the
Care and Use of Laboratory Animals (7th edition, United States).
To evaluate the anti-influenza virus effects of epigoitrin on
mice loaded with restraint stress, mice were randomly distributed
to six groups: Control, Virus, “Restraint +Virus,” Oseltamivir
(30 mg/kg/d oseltamivir +restraint +virus), Epigoitrin-L
(88 mg/kg/d epigoitrin +restraint +virus), and Epigoitrin-H
(176 mg/kg/d epigoitrin +restraint +virus). Oseltamivir and
epigoitrin were administered orally to mice for 7 consecutive
days, while other groups were received oral administration of
water only. After the first day of administration, mice except
those in Control and Virus groups were physically restricted in
the plastic centrifuge tube of 50 mL with holes for 22 h. On the
second day after restraint, mice were anesthetized by inhalation
of diethyl ether vapor and then were inoculated intranasally
with 500 PFU Influenza virus in PBS. Subsequently, the daily
changes of mice in survival and their typical influenza symptoms,
including hunched back, ruffled fur, altered respiration and
unresponsiveness, were observed and recorded for 21 days or
until death. The morbidity of the mouse was estimated when its
weight was decreased over 1 g·d−1. The survival rate was also
calculated.
Mice were weighed and euthanized after 5 days post infection
(dpi), and the lungs were removed and weighed. The lung
index was calculated according to the formula: Lung index
(mg/g) = lung weight/body weight. Samples of lung tissue were
reserved for histopathological examination, virus titers, and
western blotting analysis.
The second animal experiment was conducted to investigate
the effect and mechanism of epigoitrin on type I IFN secretion
in stressed mice. Mice were distributed at random to five
groups: Control, Virus, “Restraint+Virus,” Epigoitrin-L, and
Epigoitrin-H. The following treatment was the same as described
above. The lung tissues were collected to determine the protein
expressions related with IFN-βand MAVS signaling. To explore
the effects of epigoitrin on corticosterone level, Mice were
randomly divided into four groups: Virus, “Restraint+H1N1,”
Epigoitrin-L and Epigoitrin-H. On the second day after
restraint, mice were challenged with virus. Blood samples were
collected from the heart to determine the plasma corticosterone
levels. For investigating the anti-viral activity against H1N1
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in unstressed mice, Mice were orally administered with
epigoitrin-L (88 mg/kg/d epigoitrin +virus) and epigoitrin-
H (176 mg/kg/d epigoitrin +virus) for 7 days prior to
H1N1 infection. After 5 dpi, Lung samples were collected for
TCID50 assay.
Histological Analysis of Lung Injury
The lungs of the mice were fixed in 10% neutral buffered formalin
and processed routinely. Paraffin sections, 5–10 µm thick, were
stained with hematoxylin and eosin and then examined under
microscopy in a blinded fashion. Pathological changes were
scored based on the criteria (Fang et al., 2011): 0, no pneumonia;
1, mild interstitial pneumonia (<25% of the lung); 2, moderate
interstitial pneumonia (25–50% of the lung); 3, severe interstitial
pneumonia (>50% of the lung). The sums of scores of different
animals were averaged.
Quantification of Cells and Measurement
of Cytokines From Bronchoalveolar
Lavage Fluid (BALF)
Mice were anesthetized after 5 dpi and lungs were lavaged by
instillation and withdrawal of 1 ml PBS through a tracheal
cannula and BAL fluid (BALF) was collected. Total BAL
cell numbers were determined using a hemocytometer. After
centrifuged at 1500 rpm at 4◦C for 5 min, Supernatants were
collected to determine the levels of TNF-αand IL-1βusing
enzyme-linked immunosorbent assay (ELISA) kits (Thermo
Fisher Scientific, Waltham, MA, United States) according
FIGURE 1 | Epigoitrin attenuated the morbidity and mortality caused by influenza infection in stressed mice. (A,B) Healthy curve and morbidity caused by influenza
infection in restraint mice. (C,D) Survival curve and mortality caused by influenza infection in restraint mice. “W/O epigoitrin” indicates without epigoitrin treatment.
Epigoitrin-H and Epigoitrin-L, respectively, represent the higher dose of epigoitrin (176 mg/kg/d) and the lower dose of epigoitrin (88 mg/kg/d). The difference was
considered statistically significant at ∗P<0.05 vs. H1N1 group; #P<0.05 and ##P<0.01 vs. “Stress+H1N1” group. Data were obtained from 10–14 animals in
each group.
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FIGURE 2 | Epigoitrin protected against pneumonia caused by influenza infection in stressed mice. (A,B) The effects of epigoitrin on the viral titres in the lungs (n= 4)
and the lung index (n= 6). (C,D) Lung pathological scores (n= 3) and histopathologic changes on the 5th day after influenza virus challenge, stained by H&E (scale
bar = 50 µm). (E,F) Effects of epigoitrin on the changes of types of infiltrated inflammatory cells and cell numbers in BALF (scale bar = 100 µm). (G) Effects of
epigoitrin on the levels of TNF-α, IL-1βin BALF (n= 4). The difference was considered statistically significant at ∗P<0.05, ∗∗P<0.01, ∗ ∗ ∗P<0.001 vs. H1N1
group; #P<0.05, ##P<0.01, ### P<0.001 vs. “Stress+H1N1” group.
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to the manufacturer’s protocol, and compared with known
standards. Cell pellets were resuspended in 200 µl PBS and
cellular infiltration was assessed on Wright-Giemsa-stained slides
(Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
Determination of Corticosterone Level in
Plasma
Corticosterone was extracted from the plasma and quantified by
HPLC as we previously reported (Chen et al., 2017). Briefly, the
plasma was collected from blood samples pretreated with 10 µl
of heparin after centrifugation at 2500 ×gfor 10 min. Cortisol
solution (100 µl, 500 ng/ml) as an internal standard was mixed
into plasma (1 ml), and then were extracted by mixing with 2 ml
of ethyl acetate thoroughly for three times. The organic phase was
collected, washed, and evaporated under nitrogen. The residue
was dissolved and analyzed by HPLC with a UV detection at
254 nm (Agilent 1200).
Cell Culture and Treatment
Human alveolar epithelial cell line (A549) and Madin-Darby
canine kidney (MDCK) cells were grown at 37◦C in 5% CO2
atmosphere in Dulbecco’s modified Eagle’s medium (DMEM;
high glucose, with L-glutamine) supplemented with 10% fetal
bovine serum (FBS), 100 IU/ml penicillin, and 100 µg/ml
streptomycin. For simulating the stress-induced susceptibility to
influenza virus infection in vitro, corticosterone, an indicator of
the stress response, was employed to establish a “Corticosterone
+Virus” A549 cell model. Cells were treated with corticosterone
(100 µM) for 48 h and then infected with 10 TCID50 for
12 h. Based on the “Corticosterone +Virus” model, we then
evaluate the antiviral activity of epigoitrin against IAVs. Cells
pretreated for 2 h with different concentrations of epigoitrin
were challenged with corticosterone for 48 h in the presence
of epigoitrin prior to infection, then was infected with H1N1
influenza virus of 10 TCID50 for 1.5 h. Twelve hours post
infection (hpi), the cells were harvested for RT-qPCR and
TCID50 assay.
Viral RNA Quantification by RT-qPCR
Total RNA was extracted using TRIzol reagent (Invitrogen) at
indicated time according to manufacturer’s instructions. RNA
concentrations were determined by optical density measurement
at 260 nm on a spectrophotometer (Thermo Fisher Scientific)
and cDNA was synthesized from the purified RNA by both
random and oligo (dT) priming using an iScript cDNA synthesis
kit (Bio-Rad). Intracellular NP and IFN-βRNA levels were
measured using the SYBR green method (Applied Biosystems)
on a reverse transcription (RT) machine (CFX ConnectTM;
Applied Biosystems) and the relative values of Actin. The
fold induction of viral RNA or innate immune genes over the
levels of induction for either mock-infected cells or DMSO-
treated control cells was calculated. Primer sequences were as
follows: NP forward, 50-CAGGTACTGGGCCATAAGGAC-30,
and reverse, 50-GCATTGTCTCCGAAGAAATAAG-30; IFN-β
forward, 50-CTTACAGGTTACCTCCGAAACTGAA-30, and
reverse, 50-TTGAAGAATGCTTGAAGCAATTGT-30; Actin
forward, 50-TGACGTGGACATCCGCAAAG-30, and reverse,
50-CTGGAAGGTGGACAGCGAGG-30.
TCID50 Assay
Briefly, a confluent monolayer of MDCK cells grown in 96-well
plates were washed two times with PBS and then inoculated with
FIGURE 3 | Epigoitrin improved MAVS antiviral signaling after influenza
infection in stressed mice. (A) Effects of epigoitrin on MFN2, MAVS, IFN-β,
and IFITM3 protein expressions in the lung tissues. (B) Effects of epigoitrin on
corticosterone level in the plasma. (C) Effects of epigoitrin on MFN2, NP
protein expressions in the lung tissues. Actin was used as an internal control
and the relative densities of the measured protein were quantified by image J
software. ∗∗∗P<0.001 vs. H1N1 group; ns, no significance (P≥0.05) vs.
Stress+H1N1 group.
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threefold serial dilution of the virus-containing supernatants in
DMEM for 2 h. The inoculum was removed, and cells washed
and incubated with 200 µL DMEM containing 1 µg/mL TPCK-
trypsin. After 48 h of incubation, the cytopathic effect was scored
microscopically, and the TCID50 dose was calculated according
to the Reed–Muench methods.
Immunofluorescence Assay (IFA)
Cells from each group were fixed with 4% paraformaldehyde for
30 min, and then were permeabilized with 0.1% Triton-X100
for 5 min. After blocking with 2% bovine serum albumin for
20 min, they stained with a rabbit polyclonal antibody against NP
(GTX125989) or a mouse Monoclonal antibody against MFN2
(ab56889, Abcam, United States) in 1:100 dilution at 4◦C for
12 h. The secondary antibodies conjugated with Alexa Fluor
488 anti-mouse IgG in 1:200 dilution or 555 conjugated goat-
rabbit IgG (Life Technology, NY, United States) in 1:200 dilution
were applied for 45 min at room temperature. Nuclei were
counterstained with DAPI (Beyotime, Shanghai, China), and the
cells were visualized and analyzed using a confocal laser scanning
microscope.
Lung Histopathology and Virus Titers
Lungs from each group of mice after 5 dpi were removed and
immediately fixed in 4% buffered formalin. Subsequently, the
lung tissue was embedded in paraffin wax and 4 µm thick sections
were sliced and stained with hematoxylin and eosin (H&E). For
determination of virus titer in lung, lung tissues from euthanized
mice were aseptically removed and homogenized in DMEM
plus antibiotics to achieve 10% (w/v) suspensions, followed by
centrifuged at 1400 gfor 20 min at 4◦C. TCID50 assay was
performed to determine the infectivity of virus in the supernatant
as described above.
EGFP-MFN2 Plasmid Transfection
A549 cells were cultured on 6-well plate (NEST Biotech) in 50000
cells/ml and subsequently prepared for EGFP-MFN2 (TransGen
Biotech) transfection to reach 50–60% confluence. A non-
targeting vector (TransGen Biotech) was used as negative control.
The transfection, using Lipofectamine R
LTX & PLUSTM Reagent
(Invitrogen), was carried out as described in the instructions of
the manufacturer. Forty eight hours after transfection, cells were
infected with 10 TCID50 H1N1 for 2 h. Subsequently, cells were
performed to western blotting analysis at 12 hpi.
Western Blotting Analysis
For immunoblotting analysis, lung samples and cell lysates
lysed by RIPA buffer (Beyotime, China) were resolved by
SDS–PAGE and transferred to the polyvinylidene fluoride
membrane (Millipore, United States). Immunoblots were
visualized by the ECL system (Fdbio Science, China). The
following antibodies were used in immunoblotting analysis:
antibodies for anti-IFITM3 (1:2,000), anti-MFN2 (1:2,000),
anti-IFN-β(1:500) antibody and anti-IL-1β(1:1,000) were from
Abcam. β-actin antibody (1:2,000) was purchased from Fude
Biotechnology. Anti-phospho-IRF3 (1:1,000), and anti-TNF-
α(1:2,000) antibodies were purchased from Cell Signaling
Technology. Anti-MAVS (1:500) antibodies were obtained from
Proteintech Group. β-actin was used as an internal control and
the relative densities of the measured protein were quantified by
image J software.
Statistical Analysis
All data are expressed as mean ±standard deviation (SD)
from at least three independent experiments. Differences during
experiments were analyzed by unpaired one-way ANOVA (Tukey
test) of the GraphPad Prism 5 system. Kinetics of mortality and
morbidity are analyzed by Kaplan–Meier curves and log-rank test
with Bonferroni adjustment. A value of P<0.05 was defined as
statistically significant.
RESULTS
Protective Effects of Epigoitrin on
Influenza Infection in Restraint-Stress
Mice
To establish a susceptible animal model, mice were loaded with
restraint stress for 22 h before H1N1 virus challenge were
employed. The experimental mice were monitored daily for
21 days. Compared with “H1N1” group, the emergence of clinical
symptoms, including lack of appetite, inactivity, ruffled fur, a
hunched posture and respiratory distress had an earlier tendency
in “Restraint stress +H1N1” group (Figure 1A). Moreover,
the morbidity of “Restraint stress +H1N1” group increased to
100% (Figure 1B). These results suggested that restraint stress
could exacerbate clinical development of influenza disease and
make the host susceptible to the influenza virus. Based on
this model, the protective effects of epigoitrin against H1N1
virus in mice were investigated. As shown in Figures 1C,D,
mice in the “Restraint stress +H1N1” group had a higher
morbidity rate (100% vs. 83%) and lower mean time to sickness
(6.33 ±0.89 vs. 8.43 ±5.36 days) compared to the “H1N1”
alone group. Likewise, the survival rate of “Restraint stress +
H1N1” group decreased from 71 to 50.0% compared with the
“H1N1” alone group (P<0.01), and the mean day to death
(MDD) decreased from 17.29 ±6.16 to 10.86 ±5.7 days
(P<0.05). However, the administration of epigoitrin at 176,
88 mg/kg/d saved 50 and 29% of “Restraint stress +H1N1”
group, respectively, and prolonged MDD with a lower morbidity
sign. As a positive control, oseltamivir remarkable decreased the
morbidity rate to 38% and markedly raised the survival rate
to 92%. These results demonstrated that epigoitrin treatment
effectively increased survival rate and protected mice from lethal
infection with influenza.
Effects of Epigoitrin on Influenza
Infection in the Lung of Restraint-Stress
Mice
To further evaluate the therapeutic efficacy of epigoitrin against
influenza infection in stressed mice, virus titers in the lung were
measured after 5 dpi. No virus was detected in lung tissues of
the Control group. The virus titer in “Restraint stress +H1N1”
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group was significantly higher than that in “H1N1” alone group
(4.35 ±0.50 vs. 2.40 ±0.39 Log10 TCID50/ml), whereas virus
titer were 2.55 ±0.25 Log10 TCID50/ml and 4.13 ±0.37 Log10
TCID50/ml in the high- and low-dose epigoitrin-treated groups
(Figure 2A), respectively. H1N1 virus titers in stressed mice
after epigoitrin treatment markedly decreased. Histopathological
examination by H&E staining was performed to investigate
pathological changes after virus infection. As shown in Figure 2C,
mild inflammatory cell infiltration was observed in the lungs
of mice infected H1N1 virus. More severe inflammatory cell
infiltration in the interstitium and alveoli (Figure 2D) were
found in the “Restraint stress +H1N1” group. Meanwhile, a
higher lung index and histopathological score presented in the
“Restraint stress +H1N1” group (Figures 2B,C), compared to
the “Virus” alone group. In contrast, treatment with oseltamivir,
or epigoitrin-H significantly reduced the numbers of total cell
and infiltration of neutrophils, monocytes and lymphocytes in
BALF (Figures 2E,F) with a lower index and histopathological
score. Moreover, epigoitrin treatment effectively lowered TNF-α
and IL-1βlevels in BALF from “Restraint stress +H1N1” group
(Figure 2G).
Epigoitrin Improved the MAVS Antiviral
Signaling Pathway in Restraint Stressed
Mice
Previous studies had suggested that restraint stress-induced
influenza viral susceptibility was closely related to innate
immunity (Glaser and Kiecolt-Glaser, 2005). After infection,
H1N1 virus mRNA was recognized by RIG-I/MAVS/IRF3
pathway and induced type I IFNs secretion, which could suppress
viral replication, boost adaptive immunity, and limit acute
lung injury (Arimori et al., 2013;McNab et al., 2015). To
confirm that the differences in virus pathogenicity between
the “Restraint stress +Virus” and epigoitrin groups were due
to an improvement in type I IFNs pathway, the expression
FIGURE 4 | Epigoitrin reduced H1N1 viral expression in corticosterone-loaded A549 cell. (A) A549 cells were treated with or without corticosterone (100 µM) for
48 h before viral infection and the NP gene expression were measured by RT-qPCR at the indicated time. (B,C) Effects of epigoitrin on the NP gene expression in
H1N1-infected A549 cell pretreated with or without corticosterone. (D) Effects of epigoitrin on the viral titers of lungs in stressed or unstressed mice (n= 4). The
difference was considered statistically significant at ∗∗P<0.01, ∗ ∗ ∗P<0.001 vs. H1N1 group; #P<0.05 vs. corticosterone +H1N1 group; ns, no significance
(P≥0.05) vs. H1N1 group.
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levels of protein related to this pathway were evaluated.
The protein expressions of MAVS, IFN-βand IFITM3 were
assayed in the lung tissue. Large increases in the protein
expressions of MAVS, IFN-β, and IFITM3 were observed upon
viral infection. Compared to the “H1N1” group, restraint
stress obviously reduced these protein expressions with a
higher MFN2 level. However, this process was improved by
epigoitrin treatment (Figure 3A). Stressors could activate the
hypothalamic-pituitary-adrenal axis and thereby trigger increases
in stress hormone levels, which lead to dysregulation of immune
function. Our previous study revealed that restraint stress-
induced influenza viral susceptibility was specifically associated
FIGURE 5 | Epigoitrin inhibited H1N1 replication and promoted IFN-βgeneration after influenza infection in stress cell model. (A) Cells stained for influenza A virus
NP at 12 hpi (red) and the cell nuclei were stained by DAPI (blue). Bar = 50 µm. (B) The total fluorescent intensity was determined to reflect the levels of NP.
(C) Related infectious viral titer was detected by TCID50 assay. (D,E) Effects of epigoitrin on IFN-βgene and protein expression were analyzed by qRT-PCR and
Western blotting. The difference was considered statistically significant at ∗∗P<0.01, ∗ ∗ ∗P<0.001 vs. H1N1 group; #P<0.05, ## P<0.01, ###P<0.001 vs.
corticosterone +H1N1 group.
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with corticosterone secretion (Chen et al., 2017). Hence, effects
of epigoitrin on corticosterone level in the plasma of stressed
mice were investigated. As show in Figures 3B,C, restraint
stress significantly elevated the plasma corticosterone levels
and the expression of MFN2 and virus nucleoprotein (NP)
in lungs of mice. Interestingly, Epigoitrin-H and Epigoitrin-
L had no significant effects on the plasma corticosterone
level, but decreased the protein level of MFN2 and NP
in stressed mice. It could be inferred that restraint stress-
induced influenza viral susceptibility might be associated with
FIGURE 6 | MFN2 was involved in the regulation of IFN-βproduction by epigoitrin in stress cell model. (A) Effects of EGFP-MFN2 overexpression on H1N1 infection.
(B) The levels of protein expression by the MFN2, MAVS, p-IRF3, and IFITM3 gene relative to the level of β-actin expression were determined using the respective
antibodies. (C) Representative fluorescence images showing the levels of MFN2 protein expression. Bar = 20 µm.
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corticosterone secretion. Epigoitrin treatment would boost
resistance to influenza through the mitochondrial antiviral
signaling pathway independent of inhibiting corticosterone
secretion.
Epigoitrin Reduced H1N1 Viral
Expression in Corticosterone-Loaded
A549 Cell
According to the results of animal experiments (Figure 3B),
restraint stress-induced influenza viral susceptibility was
associated with corticosterone secretion. Thus, corticosterone
was utilized to establish a stress-induced susceptibility model
in vitro. The gene expression of NP was assessed to reflect the
viral replication level. As shown in Figure 4A, the intracellular
viral abundance in the cells treated with corticosterone and
H1N1 was significantly higher than that in cells only treated
with virus at 12 and 24 hpi. The susceptibility to H1N1 infection
caused by corticosterone is independent of viral entry, because
there was no statistically significant difference in NP gene
expression between the two groups level at 1 hpi. Based on
this model, we examined the antiviral effects of epigoitrin.
A dose-dependent inhibition in the mRNA expression of NP
was observed in cells pretreated by epigoitrin compared with
levels in untreated cells after corticosterone load (Figure 4B).
However, this preventive effect of epigoitrin on NP gene
expression in H1N1 susceptibility model may be not due to
its direct antiviral ability, because epigoitrin pretreatment had
no significantly inhibitory effect on the expression of viral
gene NP in A549 cell (Figure 4C) and virus titer in the lungs
of unstressed mice after H1N1 infection (Figure 4D). These
result were consistent with the fact that pre-treatment with
epigoitrin didn’t exert a prophylaxis effect on H1N1 infection
(Xiao et al., 2016).
Epigoitrin Inhibited H1N1 Replication
Through Promoting IFN-βGeneration in
Corticosterone-Induced Viral
Susceptibility Model
To further investigate the protective effect of epigoitrin on
reducing influenza susceptibility in vitro, NP protein levels
and virus titers were measured. As shown in Figure 5A, no
immunofluorescence of viral NP was found in the “Control”
and “Corticosterone” group. A significant increase of red
fluorescence intensity (Figure 5B) and virus titer (Figure 5C)
were observed in H1N1-infected cells with corticosterone
pretreatment, which could be attenuated by epigoitrin treatment.
Moreover, in cells pretreated with corticosterone, the expression
levels of H1N1-triggered IFN-βgene (Figure 5D) and protein
FIGURE 7 | Schematic diagram of the mechanism of Epigoitrin-induced attenuation of H1N1 pathogenesis in the susceptible model.
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Luo et al. Epigoitrin, Stress-Induced Viral Susceptibility
(Figure 5E) were significantly reduced, while epigoitrin
treatment improved their production and reduced viral
NP protein.
MFN2 Was Engaged in the Regulation of
IFN-βProduction by Epigoitrin in
Corticosterone-Induced Viral
Susceptibility Model
The antiviral RIG-I-like Receptor (RLR) signaling played a
key role for IFN-βproduction in the mammalian immune
response during H1N1 infection. Accumulated findings had
unveiled that mitochondrial dynamics participated in RLR
signaling transduction, functioning as signaling platforms
and contributing to effector responses (West et al., 2011).
MFN2, a mitofusin protein, had been shown to interact
with MAVS and suppressed MAVS activating the IFN-β
generation (Yasukawa et al., 2009). To examine the effects
of MFN2 overexpression on H1N1 infection, we transfected
cells with EGFP-MFN2 for western blot analysis. H1N1-
infected cells showed higher IFN-β, an indicator of RLR
signaling activation, and lower NP production when compared
to cells transfected with MFN2. Moreover, higher MAVS
expression was also noted in H1N1-infected cells without
MFN2 transfection while lower antiviral activity appeared
in the MFN2-overexpressing cells (Figure 6A). Hence, to
explore whether epigoitrin could down-regulate the level of
MFN2 protein and improved the MAVS antiviral signaling
in H1N1 susceptibility model. Increased MFN2 protein was
detected in both “Corticosterone” and “Corticosterone +Virus”
groups, which resulted in a decreased level of the MAVS
antiviral signaling-related proteins, MAVS, p-IRF3, and IFITM3.
However, epigoitrin treatment could reduce the MFN2 protein
expression and improve MAVS antiviral signaling-related
proteins production (Figure 6B). We also determined the effect
of epigoitrin on MFN2 expression by immunofluorescence
and the results were coincided with that from western blotting
(Figure 6C).
DISCUSSION
Previously, we had utilized restraint stress, a commonly
used stressor to establish mouse H1N1 susceptibility
model and evaluate the anti-viral effect of medicines and
compounds. Based on this model the protective effect of
epigoitrin on influenza susceptibility in vivo was evaluated.
Our results showed that substantially increased morbidity
and mortality in restraint stress animals challenged with
H1N1 virus. Moreover, restraint stress also significantly
increased the virus titer and induced excessive production
of pro-inflammatory cytokines such as IL-1β, TNF-α, which
aggravated pathological changes of the lung tissues in H1N1-
infected mice. In comparison, treatment with epigoitrin
prior to infection led to improvement of these pathological
indicators and reduced the risk of influenza virus infection in
restraint-stressed mice.
The principal peripheral effectors of the stress system are
glucocorticoids, which are regulated by the hypothalamic–
pituitary–adrenal axis (Chrousos, 2009). Studies indicated
that restraint stress-induced influenza viral susceptibility
was associated with corticosterone secretion (Konstantinos
and Sheridan, 2001;Chen et al., 2017). Thus, we utilized
corticosterone to establish an A549 cell stress model, and
evaluated the preventive effects of epigoitrin on H1N1 infection.
In the present study, we found that corticosterone in vitro could
disrupt the interferon innate immune pathways and increased
influenza viral susceptibility, which eventually facilitated its own
replication in host cells. This effect of corticosterone-induced
immunosuppression on virus infection is improved by epigoitrin.
Epigoitrin treatment could improve the MAVS antiviral signaling
and promoted the generation of IFN-βin corticosterone-loaded
A549 cells and stressed mice following H1N1 infection.
Mitochondria were essential for triggering the cellular innate
immune responses via MAVS against invading viruses, especially
RNA viruses. This subsequently activated a signaling cascade
that resulted in the phosphorylation and nuclear translocation
of IRF3, leading to the expression of type I IFN (Iwasaki
and Pillai, 2014). MFN2 acted as an inhibitor in regulating
MAVS signaling independent of its function in mitochondrial
fusion (Yasukawa et al., 2009). In accordance with this report,
we also observed that overexpressed MFN2 dampened the
generation of IFN-βfollowing viral infection and increased
H1N1 replication. The expression of MFN2 was considerably
increased under restraint stress and corticosterone load, which
was reduced by epigoitrin to improve the activation of MAVS
antiviral signaling. These data indicated that epigoitrin could
improve the suppression of innate immunity by restraint stress
via downregulating MFN2 expression (Figure 7). Nevertheless,
we do not know the underlying mechanism, further studies are
required to investigate it.
In summary, our results demonstrated that epigoitrin reduced
the susceptibility to H1N1 virus and the production of pro-
inflammatory cytokines to alleviate pneumonia in restraint-
stressed mice. Based on both the restraint-stressed mice model
and corticosterone-loaded A549 cell model, epigoitrin was found
to maintain MAVS antiviral signaling following H1N1 infection
to ensure IFN-βproduction.
AUTHOR CONTRIBUTIONS
R-RH and Y-FL developed the study design and revised the
manuscript. ZL participated in the study design, performed the
experiments, analyzed the data, and wrote the manuscript. L-FL,
X-HW, CJ, HC, D-HL, C-YY performed the experiments and
analyzed the data. BL, F-QW, and HK participated in the study
design and analyzed the data. All authors have read and approved
the final version of the manuscript.
FUNDING
This work was supported, in part, by the National Natural
Science Foundation of China (Grant Numbers 81622050,
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Luo et al. Epigoitrin, Stress-Induced Viral Susceptibility
81673709, 81560661, 81573675 and 81873209), the Young Top-
notch Talent Support Program of Guangdong Province (Grant
Numbers 2014TQ01R229 and 2016TQ03R586), the Science
and Technology Program of Guangzhou (Grant Numbers
201604046016 and 201610010182), Guangdong Science and
Technology Foundation for Distinguished Young Scholars
(2017A030306004), and the National Key Research and
Development Program of China (2017YFC1700404).
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Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest.
Copyright © 2019 Luo, Liu, Wang, Li, Jie, Chen, Wei, Lu, Yan, Liu, Kurihara, Li
and He. This is an open-access article distributed under the terms of the Creative
Commons Attribution License (CC BY). The use, distribution or reproduction in
other forums is permitted, provided the original author(s) and the copyright owner(s)
are credited and that the original publication in this journal is cited, in accordance
with accepted academic practice. No use, distribution or reproduction is permitted
which does not comply with these terms.
Frontiers in Pharmacology | www.frontiersin.org 12 February 2019 | Volume 10 | Article 78