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 c
Trang 1virus by suppression of manganese superoxide
dismutase expression
Dachun Yao1,2, Huawen Li3, Yulan Gou1,4, Haimou Zhang5, Athanasios G Vlessidis2, Haiyan Zhou4, Nicholaos P Evmiridis2and Zhengxiang Liu1
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 suc-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 c release 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; DiOC6,3,3¢-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.
Trang 2including 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 (O2·)) 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 expressuppres-sion 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
Trang 396
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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)1BetA 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)1group (D–H) Cells were treated with 15 lgÆmL)1BetA 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.
Trang 4BetA-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, DWm) 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
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Fig 2 BetA-mediated selective SOD2 suppression in HBV-infected hepatocytes The 80% confluent WT or HBV-infected cells were treated with 15 lgÆmL)1BetA 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.
Trang 5possible 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
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TGACGTCT
CREB
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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)1BetA, 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.
Trang 6protein level (Fig 4E) The results showed that both
SOD2 protein expression and mRNA expression
were decreased after BetA treatment, and that this
effect was abolished by CREB overexpression in the
presence of BetA, but was mimicked by siCREB
This indicates that SOD2 expression is regulated by
CREB phosphorylation at Ser133 As we had
already shown that BetA treatment decreased CREB
phosphorylation at Ser133 (Fig 4C), we next per-formed in vitro experiments to determine whether BetA could inhibit CREB phosphorylation directly
As shown in Fig 4F, the purified CREB was sub-stantially phosphorylated at Ser133 in the presence
of PKA, whereas phosphorylation was markedly inhibited by BetA, indicating that BetA could directly inhibit CREB phosphorylation, and this
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Fig 4 BetA-mediated SOD2 suppression was due to direct inhibition of CREB phosphorylation (A) HBV-infected hepatocytes were treated with control (CTL), 15 lgÆmL)1BetA, BetA with CREB overexpression (BetA ⁄ CREB›) or siRNA for CREB (siCREB) for 48 h; the chromatin from treated cells was immunoprecipitated with CREB antibody, and the SOD2 promoter that covers the CREB-binding motif was amplified
by quantitative PCR (qPCR) (B–E) The cells treated as above were used for measurement of CREB protein level (B), pCREB protein level (C), SOD2 mRNA level (D), and SOD2 protein level (E) *P < 0.05 versus CTL for (A)–(E) (F) In vitro-purified proteins were phosphorylated
by PKA in the presence or absence of BetA, and pCREB was measured by western blotting *P < 0.05 versus first panel; –P < 0.05 versus second panel.
Trang 7decreased amount of phosphorylated CREB (or
decreased CREB activity) downregulates SOD2
expression through the CREB-binding motif on the
SOD2 promoter
BetA suppresses HBx and translocates HBx to
mitochondria
We further examined the effect of BetA on HBx from
HBV-infected hepatocytes The cells treated with
con-trol, BetA or siCREB were isolated into
mitochon-drial and cytosolic fractions for western blotting
analysis As shown in Fig 5A, the level of HBx
pro-tein was decreased in total lysates and cytosolic
frac-tions but increased in mitochondrial fracfrac-tions after
BetA treatment, and siCREB mimicked the effect of
BetA, indicating that BetA treatment not only
sup-pressed HBx expression, but also translocated HBx
into mitochondria We further measured
cyto-chrome c release for the treated cells As shown in
Fig 5B, the cytochrome c level was substantially
increased in cytosolic fractions after BetA or siCREB
treatment as compared with control, was decreased in
mitochondria, but was unchanged in total lysates
This suggests that BetA-mediated cytochrome c release
and apoptosis may be associated with HBx transloca-tion to mitochondria
The BetA-mediated proapoptotic effect depends
on BetA-induced SOD2 suppression in HBV-infected cells
In order to further determine the mechanisms of BetA-induced HBx translocation and proapoptotic effects, we measured the BetA-induced cytotoxicity in different cells; as shown in Fig 5C,D, BetA slightly increased caspase-3 activity (Fig 5C) and the apop-tosis rate (Fig 5D) in WT cells, and a similar effect was observed in WT hepatocytes overexpressing HBx; overexpression of CREB could not abolish this effect, suggesting that the BetA-induced basal toxic effect in WT cells is not due to BetA-induced SOD2 suppression, and HBx alone is not directly involved
in BetA-induced basal toxicity On the other hand, the BetA-induced toxicity was substantially increased
in HBV-infected hepatocytes as compared with WT cells, and this effect was mostly abolished by overex-pression of CREB, suggesting that, in HBV-infected cells, BetA-induced toxicity is due to SOD suppres-sion We next measured SOD2 expression in different
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Fig 5 BetA reduces the level of HBx and translocates HBx to mitochondria through SOD2 suppression and subsequent ROS generation (A, B) HBV-infected hepatocytes were treated with 15 lgÆmL)1BetA for 48 h, the cells were separated as mitochondrial and cytosolic frac-tions, and the protein levels were measured by western blotting (A) HBx protein (B) Cytochrome c (CytC) protein *P < 0.05 versus control (CTL) group (C–E) WT hepatocytes, WT cells overexpressing HBX (WT-HBx cells) or HBV-infected hepatocytes were treated with either CTL, 15 lgÆmL)1BetA or BetA with CREB overexpression (BetA ⁄ CREB›) for 48 h (C) Intracellular caspase-3 activity *P < 0.05 versus CTL; –P < 0.05 versus BetA in the HBV-infected group (D) Apoptosis rate determined by TUNEL assay *P < 0.05 versus CTL; –P < 0.05 versus BetA in the infected group (E) SOD2 mRNA level *P < 0.05 versus CTL in the WT group (F) The mitochondrial fraction from the HBV-infected hepatocytes treated as above was used for analysis of HBx by western blotting The WT HBx group shows no detectable bands in mitochondria (data not shown) *P < 0.05 versus CTL.
Trang 8cells; as shown in Fig 5E, the basal level of SOD2
was not changed in WT cells or WT hepatocytes
overexpressing HBx, whereas the SOD2 level was
substantially increased in HBV-infected cells as
com-pared with WT cells; this increase was totally
nor-malized by BetA, and overexpression of CREB
minimized the effect of BetA, suggesting that
BetA-induced toxicity in HBV-infected cells is due to
BetA-mediated SOD suppression Finally, we
mea-sured HBx translocation to mitochondria in different
cells, as shown in Fig 5F In WT hepatocytes
over-expressing HBx, HBx was not found in mitochondria
at all in the presence of BetA (data not shown),
whereas in HBV-infected cells, BetA-induced HBx
translocation was totally abolished by CREB
overex-pression, suggesting that BetA-induced SOD2
suppression and subsequent ROS generation is the
driving force for HBx transcloation to mitochondria
The BetA-mediated inhibitory effect on HBV is due to SOD2 suppression and subsequent ROS generation
We previously found that BetA suppresses SOD2 expression by inhibiting CREB phosphorylation, with subsequent ROS overgeneration Here, we further investigated the potential effect of SOD2 on HBV replication The HBV-infected hepatocytes were trea-ted with BetA, or BetA with SOD2 overexpression,
or siRNA for SOD2 (siSOD2) alone, and the related biomedical parameters were measured As shown in
Fig 6, the levels of SOD2 mRNA (Fig 6A) and SOD2 protein (Fig 6B) decreased to 33% and 46%, respectively, after BetA treatment, BetA treatment with SOD2 overexpression caused no difference in SOD2 level, and siSOD2 mimicked the effect of BetA We also measured ROS formation (Fig 6C)
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Fig 6 BetA-mediated inhibitory effect on HBV through SOD2 suppression and ROS generation HBV-infected hepatocytes were treated with control (CTL), 15 lgÆmL)1BetA, BetA with CREB overexpression (BetA ⁄ -CREB›) or siCREB for 48 h, and the cells were used for measurement of the indicated parameters (A) SOD2 mRNA level (B) SOD2 protein level (C) ROS generation (D) Apopto-sis rate determined by TUNEL assay (E) HBsAg secreted from cell culture medium (F) HBeAg secreted from cell culture med-ium (G) HBV DNA from treated cells was measured by real-time quantitative PCR (H) HBx protein level was measured by western blotting and quantitated *P < 0.05 versus the CTL group.
Trang 9and apoptosis (Fig 6D); BetA treatment substantially
increased ROS generation and the apoptosis rate,
SOD2 overexpression in the presence of BetA
mini-mized the effect, and siSOD2 mimicked the effect of
BetA We next measured the effect of these
treat-ments with different SOD2 expression levels on HBV
replication; the results showed that BetA alone
sub-stantially inhibited HBV replication, including
hepati-tis B surface antigen level (HBsAg) level (Fig 6E),
hepatitis B external core antigen (HBeAg) level
(Fig 6F), HBV DNA (Fig 6G), and HBx protein
expression (Fig 6H), whereas a combination of BetA
and SOD2 overexpression totally abolished the
BetA-mediated HBV-inhibitory effect On the other hand,
SOD2 knockdown (siSOD2) mimicked the
induced inhibitory effect This suggests that
BetA-induced ROS generation plays an important role in
HBV inhibition; scavenging of ROS by
overexpres-sion of the antioxidant enzyme SOD2 might not
be beneficial, but worsen the HBV infection, whereas
the increase in ROS generation caused by direct
SOD2 knockdown could achieve similar
HBV-inhibi-tory effects
BetA mimics the PC-induced inhibitory effect on
HBV in mice
In order to verify that BetA or PC extract does not
alter general liver function and has no toxic effects
in healthy liver, nontransgenic mice were employed
to evaluate the proapoptotic effect Thirty male
non-HBV-transgenic mice were randomly separated into
three groups (10 in each) Experimental groups
received either purified BetA (2 mgÆkg)1) or PC
crude extracts (50 mgÆkg)1), whereas the control
group received only vehicle Drugs or vehicle were
added to the normal food and mixed for feeding
After 3 months, mice were killed by decapitation
The liver tissues were collected for measurement of
biomedical parameters: (a) SOD2 mRNA level; (b)
enzymatic activities of caspase-3 and SOD2; (c)
superoxide release; and (d) enzymatic activities of
alanine aminotranferase and aspartate
aminotrans-ferase We found that neither BetA nor PC extract
had significant cytotoxic effects on hepatocytes from
mice (data not shown) In addition, we have
previ-ously found that BetA isolated from PC inhibits
HBV replication in vitro by SOD2 suppression, which
is similar to the effect that PC had in hepatitis B
patients in our preliminary observation (data not
shown) Here, we used HBV-transgenic mice to
determine whether BetA could achieve the same
inhibitory effect As shown in Fig 7A, both BetA
and PC significantly reduced HBsAg and HBeAg serum levels and HBV DNA replication Also, both BetA and PC substantially decreased SOD2 mRNA expression, whereas CREB mRNA showed no changes (Fig 7B) In addition, protein levels of SOD2 and pCREB were substantially reduced after BetA and PC treatment, whereas no changes were found in CREB total protein level (Fig 7C) We also examined the enzymatic activities, and showed that both BetA and PC not only decreased SOD2 activity, but also increased caspase-3 activity, indicat-ing increased cytotoxicity with apoptosis rate (Fig 6D) Finally, we examined the levels of super-oxide release in different tissues (Fig 6E,F); both BetA and PC specifically increased superoxide anion generation in liver tissue, but had little effect in aorta, and no effect at all in kidney and brain, indi-cating that both BetA-mediated and PC-mediated HBV inhibition are due to specifically decreased SOD2 expression with subsequent ROS generation in liver tissue
Discussion
This study demonstrates that BetA inhibits HBV repli-cation by suppression of SOD2 expression with subse-quent mitochondrial ROS overgeneration, with promising HBV clearance in both in vitro and in vivo mouse experiments This is the first time that we have shown the potential effects and possible mechanism of HBV inhibition by BetA
BetA-mediated selective cytotoxicity in HBV-infected hepatocytes
We have found that BetA has little cytotoxic effect on
WT hepatocytes, but shows a selective cytotoxic effect
on HBV-infected hepatocytes In addition, our data showed that the basal level of SOD2 was not changed
in WT or WT hepatocytes overexpressing HBx, whereas the SOD2 level was substantially increased in HBV-infected cells as compared with WT cells, that this increase was totally normalized by BetA, and that overexpression of CREB could minimize the effect of BetA (Fig 5E), suggesting that BetA-induced toxicity
in HBV-infected cells is due to BetA-mediated SOD suppression This indicates that HBV infection in HBV-infected cells specifically increases SOD2 expres-sion, even though the detailed mechanisms are still unknown Furthermore, the basal SOD2 protein level
in HBV-infected cells is much higher than in WT cells, and it is reasonable that the HBV-infected cells with high levels of SOD2 expression should be more
Trang 10susceptible to BetA-mediated SOD2 suppression, and,
subsequently, the SOD2 suppression-mediated large
increase in mitochondrial ROS generation may further
induce mitochondrial dysfunction and apoptosis [24]
Given the fact that HBV-infected cells are more
sus-ceptible to BetA-induced SOD2 suppression, and
BetA-induced SOD2 suppression could directly inhibit
HBV replication, as shown in Fig 6, we conclude that
BetA could be a good candidate for anti-HBV drug
development
BetA-mediated CREB dephosphorylation
As BetA could cause CREB dephosphorylation at Ser133 both in vivo and in vitro, and a mutated form
of CREB with an Ala substitution for Ser133 has been reported to be a negative transcriptional regulator, BetA-induced dephosphorylation of CREB could act
as a repressor of SOD2 gene transcription directly [25] CREB, as a direct substrate of both PKA [21,26] and protein kinase D (PKD) [27], could be phosphorylated
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Fig 7 BetA-mediated HBV inhibitory effect in mice through SOD2 suppression and ROS generation HBV-transgenic mice were treated with either vehicle [control (CTL)], BetA or PC crude extracts for 3 months, the mice were killed, and the medical parameters from blood or different tissues were measured (A) HBsAg, HBeAg and HBV DNA were measured from blood (B) mRNA expression for CREB and SOD2 was measured by quantitative PCR from liver tissue (C) Protein levels for CREB, pCREB and SOD2 were measured by western blotting from liver tissue and quantitated (D) Enzyme activities for caspase-3 and SOD2 from liver tissue were measured and expressed as arbitrary units (E) Superoxide anion release from different tissues was measured (F) Representative images for in vivo superoxide staining from liver tissue *P < 0.05 versus CTL group.