Betulinic acid-mediated inhibitory effect on hepatitis B
virus by suppression of manganese superoxide
dismutase expression
Dachun Yao
1,2
, Huawen Li
3
, Yulan Gou
1,4
, Haimou Zhang
5
, Athanasios G. Vlessidis
2
, Haiyan Zhou
4
,
Nicholaos P. Evmiridis
2
and Zhengxiang Liu
1
1 Internal Medicine of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
2 Laboratory of Analytical Chemistry, Department of Chemistry, University of Ioannina, Greece
3 Department of Nutrition and Food Hygiene, Guangdong Medical College, China
4 The First Hospital of Wuhan, China
5 School of Life Sciences, Hubei University, Wuhan, China
Hepatitis B virus (HBV) infection is a prevalent health
problem, affecting 350 million people worldwide; it
causes acute and chronic hepatitis, some cases of
which may progress into cirrhosis and hepatocellular
carcinoma [1]. Chronic HBV patients are currently
treated with interferon or some nucleotide analogs,
including lamivudine and adefovir, but the poor suc-
cess and frequent recurrence after cessation of therapy
require new strategies for terminating this viral infec-
tion. Some complementary and alternative medicines,
Keywords
apoptosis; CREB; mitochondrial;
Pulsatilla chinensis; reactive oxygen species
Correspondence
D. Yao and Z. Lin, Internal Medicine of
Tongji Hospital, Tongji Medical College,
Huazhong University of Science and
Technology, Wuhan 430030, China
Fax: +86 27 83662622
Tel: +86 27 83662601
E-mail: dachun927@hotmail.com;
zxliu_tjmu@yahoo.com
(Received 3 December 2008, revised 26
February 2009, accepted 27 February 2009)
doi:10.1111/j.1742-4658.2009.06988.x
The betulinic acid (BetA) purified from Pulsatilla chinensis (PC) has been
found to have selective inhibitory effects on hepatitis B virus (HBV). In
hepatocytes from HBV-transgenic mice, we showed that BetA substantially
inhibited HBV replication by downregulation of manganese superoxide
dismutase (SOD2) expression, with subsequent reactive oxygen species gen-
eration and mitochondrial dysfunction. Also, the HBV X protein (HBx) is
suppressed and translocated into the mitochondria followed by cyto-
chrome crelease. Further investigation revealed that SOD2 expression was
suppressed by BetA-induced cAMP-response element-binding protein
dephosphorylation at Ser133, which subsequently prevented SOD2 tran-
scription through the cAMP-response element-binding protein-binding
motif on the SOD2 promoter. SOD2 overexpression abolished the inhibi-
tory effect of BetA on HBV replication, whereas SOD2 knockdown mim-
icked this effect, indicating that BetA-mediated HBV clearance was due to
modulation of the mitochondrial redox balance. This observation was fur-
ther confirmed in HBV-transgenic mice, where both BetA and PC crude
extracts suppressed SOD2 expression, with enhanced reactive oxygen
species generation in liver tissues followed by substantial HBV clearance.
We conclude that BetA from PC could be a good candidate for anti-HBV
drug development.
Abbreviations
BetA, betulinic acid; CREB, cAMP-response element-binding protein; DiOC
6,
3,3¢-dihexiloxadicarbocyanine; HBeAg, hepatitis B external core
antigen; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; HBx, hepatitis B virus X protein; MMP, mitochondrial membrane
potential; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; PC, Pulsatilla chinensis; PKA, protein kinase A; PKD, protein
kinase D; ROS, reactive oxygen species; siCREB, small interfering RNA for cAMP-response element-binding protein; siRNA, small interfering
RNA; siSOD2, small interfering RNA for manganese superoxide dismutase; SOD2, manganese superoxide dismutase; TUNEL,
deoxynucleotidyl transferase dUTP nick end labeling; WT, wild-type.
FEBS Journal 276 (2009) 2599–2614 ª2009 The Authors Journal compilation ª2009 FEBS 2599
including some herbs, have been used for centuries to
treat viral hepatitis, but they are still not widely
accepted by conventional medicine, owing to the lack
of mechanisms and purity of herbs [2].
Pulsatilla chinensis (PC) is a traditional Chinese herb
used for the treatment of amoebic diseases, vaginal
trichomoniasis, and bacterial infections, owing to its
antiamoebic, antibacterial and antitrichomonal activi-
ties [3]. Recently, this herb was used for the treatment
of a hepatitis B patient, according to an old recipe in a
specific area of China (Yichang, Hubei), with satisfac-
tory results for HBV clearance. In order to determine
the mechanism of this, about 30 components from PC
were isolated, and each of them was tested for HBV
clearance. The results revealed that the active compo-
nents were betulinic acid (BetA) and its derivatives
[4,5]. BetA, identified as a pentacyclic triterpene, is
widely available from common natural sources and
possesses several biological properties, including anti-
inflammatory, antiviral, antimalarial, and antimicro-
bial, as well as impressive anticancer and anti-HIV
activities [6–8], although the exact mechanism remains
unclear [9,10].
Manganese superoxide dismutase (SOD2) is an anti-
oxidant enzyme located in mitochondria that can scav-
enge superoxide anions (O
2
·
)
) to form hydrogen
peroxide. Suppression of SOD2 expression may lead to
the overgeneration of reactive oxygen species (ROS)
from mitochondria, and this can subsequently trigger
mitochondrial dysfunction and apoptosis. Altered
SOD2 expression is considered to be both beneficial
and detrimental. For instance, overexpression of SOD2
could be protective against ROS-mediated cell damage,
but it may also increase the invasiveness of tumors
and increase the possibility of infection [11,12]. Several
transcription factors, including specificity protein 1
and nuclear factor-jB [13,14], as well as methylation
[15,16], have been studied extensively for the regulation
of SOD2 expression, whereas there are few reports on
the role of cAMP-response element-binding protein
(CREB) in SOD2 expression [17,18]. CREB binds via
its basic leucine zipper domain as a dimer to cAMP
response elements containing the consensus motif
5¢-TGACGTCA-3¢; these are present in the promoters
of many genes in which transcription rates are strongly
regulated by cAMP. CREB stimulates cellular gene
transcription via the protein kinase A (PKA)-mediated
phosphorylation of CREB at Ser133 [19]. Ser133 phos-
phorylation of CREB, in turn, promotes recruitment
of the coactivator paralogs CREB-binding protein and
p300 via a kinase-inducible domain in CREB, which
appears to be sufficient for the induction of cellular
genes [20,21]. On the other hand, inhibition of CREB
phosphorylation or dephosphorylated CREB may be a
negative regulator of CREB-responsive genes [22,23].
In an effort to investigate the mechanism of the
inhibitory effect of BetA on HBV, BetA was isolated
from PC to treat hepatocytes from HBV-transgenic
mice. We found that SOD2 was downregulated by
BetA-induced CREB dephosphorylation at Ser133
through the CREB-binding motif on the SOD2 pro-
moter. SOD2 suppression-mediated ROS generation
subsequently inhibited HBV replication, decreased
HBV X protein (HBx) total level, and translocated
HBx to the mitochondria followed by cytochrome c
release. Overexpression of SOD2 totally abolished the
BetA-mediated HBV-inhibitory effect, whereas SOD2
knockdown mimicked this effect, indicating that the
BetA-induced HBV-inhibitory effect is due to SOD2
suppression and subsequent ROS generation. Further
in vivo experiments with HBV-transgenic mice con-
firmed our hypothesis; we found that BetA or PC
crude extracts achieved significant HBV clearance, with
decreased SOD2 expression and increased ROS genera-
tion in liver tissue. This is the first time that suppres-
sion of SOD2 expression has been found to be the
mechanism by which BetA inhibits HBV replication.
Results
BetA-induced selective cytotoxicity in
HBV-infected hepatocytes
We first examined the cytotoxicity of BetA in wild-
type (WT) and HBV-infected hepatocytes. Different
dosages of BetA were used to treat the cells for 48 h.
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazo-
lium bromide (MTT) assay results showed that there
was little effect on WT cells, whereas BetA treatment
caused significant cytotoxicity in HBV-infected cells
(Fig. 1A). Also, the time course results showed that
WT cells were more resistant to BetA-mediated cyto-
toxicity than HBV-infected cells (Fig. 1B). On the
basis of the above observation, we further evaluated
BetA-mediated cell proliferation; as shown in Fig. 1C,
HBV-infected cells showed a higher DNA synthesis
rate than WT cells with a low dose (5 lgÆmL
)1
)of
BetA, whereas with a high dose (15 lgÆmL
)1
), the
DNA synthesis rate of HBV-infected cells was sub-
stantially decreased, but WT cells showed no signifi-
cant decrease. On the other hand, when the BetA dose
was even higher (20 lgÆmL
)1
), the DNA synthesis rate
of HBV-infected cells was substantially inhibited,
whereas no difference was found in WT cells, indi-
cating that BetA-induced cytotoxicity was specific
to HBV-infected cells.
Betulinic acid inhibits hepatitis B virus D. Yao et al.
2600 FEBS Journal 276 (2009) 2599–2614 ª2009 The Authors Journal compilation ª2009 FEBS
96 84
72
60
48
36 24 12 0
2
3
3.5
A C
B D
E F
G H
2.5
1.5
2
3
3.5
2.5
1.5
30 25
20 15 10 5 0
Dosages of betulinic acid (µg·mL
–1
)
Betulinic acid exposing time (h)
Cell viability (A/10
6
cells)
Cell viability (A/10
6
cells)
WT
HBV
WT
HBV
0
300
200
100
0
300
200
100
0
300
200
100
0
300
200
100
400
300
200
100
0 µg·mL
–1
10 µg·mL
–1
15 µg·mL
–1
20 µg·mL
–1
[3H]-thymidine incorporation
(% control)
WT
WT HBV
WT HBV
WT HBV
WT HBV
WT HBV
HBV
*
*
*
*
0
ROS formation (Arbitrary units)
CTL
BetA
*
*
*
Intracellular ATP level
(Arbitrary units)
CTL
BetA
CTL
BetA
CTL
BetA
CTL
BetA
*
Relative ΔΨ
m
*
Caspase-3 activity
(pmol–1·min–1·mg–1)
*
*
0
5
20
15
10
Apoptosis rate (%)
*
*
Fig. 1. BetA-mediated selective effect on HBV-infected hepatocytes. (A) WT or HBV-infected (HBV) hepatocytes were treated with different
doses of BetA for 48 h, and cell viability was measured. (B) Cells were treated with 15 lgÆmL
)1
BetA for different times, and cell viability
was measured. (C) Cells were treated with different doses of BetA as indicated for 48 h, and then incubated with [
3
H]thymidine for 2 h to
measure the inhibitory effect of BetA on cell differentiation by the [
3
H]thymidine incorporation assay. *P< 0.05 versus WT; –P< 0.05
versus 0 lgÆmL
)1
group. (D–H) Cells were treated with 15 lgÆmL
)1
BetA for 48 h, and the related parameters were measured. (D) BetA-
induced ROS generation. (E) Intracellular ATP level. (F) MMP (Dw
m
). (G) Apoptosis rate determined by TUNEL assay. (H) Intracellular
caspase-3 activity. *P< 0.05 versus control (CTL); –P< 0.05 versus WT group.
D. Yao et al. Betulinic acid inhibits hepatitis B virus
FEBS Journal 276 (2009) 2599–2614 ª2009 The Authors Journal compilation ª2009 FEBS 2601
BetA-mediated ROS generation and
mitochondrial dysfunction was specific to
HBV-infected hepatocytes
ROS generation was then examined, and the results
are shown in Fig. 1D. BetA substantially induced ROS
generation in HBV-infected hepatocytes, as compared
with WT cells. As BetA inhibited HBV-infected cell
growth with increased ROS generation, we hypothe-
sized that BetA might also specifically affect mitochon-
drial function in those cells. Measurement of
intracellular ATP generation (Fig. 1E) revealed that
BetA treatment substantially decreased intracellular
ATP generation in HBV-infected cells, but showed no
effect on WT cells. In addition, mitochondrial mem-
brane protential (MMP, DW
m
) was substantially
decreased in HBV-infected cells, but no difference was
found in WT cells (Fig. 1F). Finally, the apoptosis
rates determined by terminal deoxynucleotidyl transfer-
ase dUTP nick end labeling (TUNEL) assay (Fig. 1G)
and caspase-3 activity (Fig. 1H) were assessed. The
results showed that BetA substantially increased the
apoptosis rate and caspase-3 activity in HBV-infected
cells as compared with WT cells.
BetA-mediated selective SOD2 suppression in
HBV-infected hepatocytes
In order to clarify the effect of BetA, a microarray
assay after treatment with 15 lgÆmL
)1
BetA for 48 h
was conducted. BetA specifically decreased SOD2
mRNA expression in HBV-infected cells, whereas little
difference was seen in WT cells (data not shown).
Real-time PCR was performed for confirmatory pur-
poses, and suggested that the SOD2 mRNA level was
decreased about 2.4-fold in HBV-infected cells trea-
ted with BetA as compared with the control, but
showed no difference in WT cells (Fig. 2A). Western
blotting to measure the protein level (Fig. 2B) showed
a significant decrease in SOD2 protein in HBV-infected
cells after BetA treatment, but no change in WT cells.
SOD2 enzyme activity (Fig. 2C) decreased significantly
in HBV-infected cells after BetA treatment, whereas
little difference was found in WT cells.
The BetA-mediated SOD2 transcriptional
response element was located at the
CREB-binding site (nucleotide )1335) on the
SOD2 promoter
The mechanism of BetA-mediated SOD2 suppression
was investigated further. To localize the regulatory
elements required for transcriptional suppression of
the SOD2 gene by BetA treatment, progressive 5¢-
promoter deletion constructs, including )2000,
)1500, )1200, )1000, )500, )200, )100, and 0, were
generated (numbered according to Ensembl Tran-
script ID: ENST00000337404). As shown in Fig. 3A,
the )2000 and )1500 constructs showed a decrease
in activity of about 55%, whereas, with other dele-
tions from )1200 to 0, the reporter activity showed
no significant decrease after BetA treatment. These
data indicate that promoter elements between )1500
and )1200 are responsible for BetA-induced tran-
scriptional suppression of the SOD2 promoter. Com-
parison of these sequences with transcription factor
databases (TFSEARCH) revealed several potential
binding motifs, including GATA ()1488), c-Ets
()1377), CREB ()1335) and NRF2 ()1247). The
0
400
A
B
C
300
200
100
0
400
300
200
100
0
400
300
200
100
WT HBV
WT HBV
WT HBV
SOD2 mRNA level by qPCR
(Arbitrary units)
CTL
BetA
CTL
BetA
CTL
BetA
*
SOD2 protein by Western blot
(Arbitrary units)
*
SOD2 protein activity
(Arbitrary units)
*
Fig. 2. BetA-mediated selective SOD2 suppression in HBV-infected
hepatocytes. The 80% confluent WT or HBV-infected cells were
treated with 15 lgÆmL
)1
BetA for 48 h, and SOD2 expression and
activity were measured. (A) mRNA level. (B) Protein level. (C)
SOD2 enzyme activity. *P< 0.05 versus control (CTL); –P< 0.05
versus WT group.
Betulinic acid inhibits hepatitis B virus D. Yao et al.
2602 FEBS Journal 276 (2009) 2599–2614 ª2009 The Authors Journal compilation ª2009 FEBS
possible involvement of these motifs in BetA-induced
SOD2 transcriptional suppression was explored using
a series of luciferase constructs with single mutations.
As shown in Fig. 3B, the SOD2 reporter with the
CREB-binding motif single mutation at )1335 from
nucleotides C to T totally abolished the BetA-
induced SOD2 suppression, whereas the mutations in
other motifs did not decrease the effect (data not
shown). This indicates that the CREB motif at
)1335 is required for BetA responsiveness of the
SOD2 promoter. As the CREB-binding motif was
localized to the BetA-responsive element, the effect
of CREB protein on SOD2 reporter activity was
examined. The SOD2 WT reporter (SOD2 )1500)
showed suppression by BetA treatment, overexpres-
sion of CREB in the presence of BetA totally abol-
ished the effect, and CREB knockdown alone [small
interfering RNA (siRNA) for CREB (siCREB)] mim-
icked this effect (Fig. 3C). On the other hand, the
SOD2 mutation reporter [SOD2 )1500 ⁄)1335(T)]
showed no effect of either BetA, overexpression of
CREB in the presence of BetA, or siCREB alone,
further demonstrating that the BetA-induced SOD2
suppression is regulated by CREB.
BetA-mediated SOD2 suppression is due to
BetA-induced CREB dephosphorylation
We have shown transcriptional activities of SOD2
that responsible to BetA treatment is due to the exis-
tence of CREB-binding elements on SOD2 promoter.
Here, we further confirmed the CREB-binding activ-
ity through chromatin immunoprecipitation analysis,
as shown in Fig. 4A. After immunoprecipitation and
reversal of the crosslinking, the endogenous SOD2
promoter was enriched by real-time PCR amplifica-
tion, using specific primers that cover the CREB-
binding motif. The results showed that the PCR
product was decreased to 47% after BetA treatment
as compared with the control group, and that the
effect was totally abolished by CREB overexpression
in the presence of BetA, whereas CREB knockdown
(siCREB) mimicked the effect. As it is well known
that CREB activity mainly depends on phosphoryla-
tion at Ser133, we next measured the levels of both
CREB protein and CREB protein phosphorylated at
Ser133 (pCREB). As shown in Fig. 4B,C, the total
CREB protein level did not change after BetA treat-
ment as compared with control, whereas the pCREB
level decreased by 42%. On the other hand, over-
expression of CREB in the presence of BetA
increased the CREB level 1.7-fold, but did not
increase the pCREB level, whereas knockdown of
CREB (siCREB) decreased the levels of both CREB
protein and pCREB. Using the above treatment, we
next measured the SOD2 mRNA level (Fig. 4D) and
0
200
100
CTL BetA si CREB
BetA / CREB
SOD2 transcriptional activity
(Arbitrary units)
SOD2–1500
SOD2–1500/ – 1335 (T)
*
*
0
120
A
B
C
100
80
60
40
20
SOD2–2000
SOD2–1500
SOD2–1200
SOD2–1000
SOD2–500
SOD2–200
SOD2–100
SOD2–0
SOD2 transcriptional activity
(Arbitrary units)
CTL
Bet A
*
*
*
#
#
*
*
*
0
BERC cuL
TCT
G
CAGT
T
CTGCAGT
TCTGTA
G
T
BERC
0
c
u
L
BERC
0
cuL
0
50
100
150
CTL BetA
–1500 –1335
–1335
–1335
–1500
–1500
SOD2–1500
SOD2–1500
Mut–1335(T)
BERC BERCCREB cuL cuL
+308
TCT
G
CAGT
T
CTGCAGT
TCTGTA
G
T
T
CTGCAGT
TCTGTA
G
T
T
CTGCAGT
TCTGTA
G
T
BERC c
u
L
BERC c
u
L
BERC BERC c
uL c
uL
Luc
+ 308
BERC
+ 308
cuL
BERC cuL
BERC BERC cuL cuL
SOD2 transcriptional activity
(Arbitrary units)
*
TGACGTCT
CREB
CREB
Luc
Luc
Fig. 3. Mapping of the BetA-responsive element on the SOD2 pro-
moter. (A) HBV-infected hepatocytes were transfected with the
indicated SOD2 reporter constructs, and then treated with either
control (CTL) or 15 lgÆmL
)1
BetA for 48 h; the SOD2 reporter
activity was then measured. *P< 0.05 versus CTL in the SOD2–
2000 group; –P< 0.05 versus CTL. (B) The above cells were
transfected with either SOD2–1500 reporter WT construct or
SOD2–1500 single mutant )1335(T); after the treatment as
indicated above, SOD2 reporter activity was measured. *P< 0.05
versus CTL. (C) HBV-infected hepatocytes were transfected with
either SOD2 )1500 or SOD2 )1500 ⁄)1335(T) single mutant
reporters, and then treated with CTL, 15 lgÆmL
)1
BetA, BetA with
CREB overexpression (BetA ⁄CREB›) or siCREB for 48 h, and
SOD2 reporter activity was measured. *P< 0.05 versus CTL in
the SOD2 )1500 group.
D. Yao et al. Betulinic acid inhibits hepatitis B virus
FEBS Journal 276 (2009) 2599–2614 ª2009 The Authors Journal compilation ª2009 FEBS 2603