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Introduction
Oenanthe javanica (Oj),
umbelliferate, has been widely used in traditional Chinese
medicine for treatment of jaundice, hypertension, and polydipsia
diseases for many years[1]. Previous studies have shown
that it has liver-protective[2], hypotensive[3],
anti-arrhythmic[4], anti-anaphylactic[5], and
antidiabetic[6] effects. Recent studies also show that
Oj was helpful in treatment of hepatitis B virus (HBV) infection
in clinical trials[7], and had an inhibitory effect on
duck hepatitis B virus (DHBV)-induced hepatitis in Nestling ducks
in vivo[8] and in vitro[9].
However, which is the active part and what are the active components
of Oj in inhibiting HBV remain unclear. Using modern
techniques, it has been revealed that OjF is one of the main
active parts against HBV and it comprises approximately 2.2% of the
whole plant content. The present study aimed to investigate the
anti-hepatitis activity of OjF in 2.2.15 cells in vitro
and duck HBV infection in a duck model in vivo.
Materials and methods
Preparation of OjF
Oj was collected from the Yanbian Autonomus Region in autumn
and identification was performed by Prof Hui-zhong XIAO, Department
of Phyto-chemistry, Yanbian Medical University. Dried whole plant
was pulverized and extracted with 80% ethanol, and subsequently
partitioned in ethyl acetate. The ethyl acetate soluble materials
were fractionated by ethanol and distilled water gradient of column
chromatography in polyamide. OjF was eluted and the content
was determined according to aluminium nitrate reagent method. The
content of hyperoside, the major ingredient of OjF, was
determined by reverse phase-high performance liquid chromatography
(RP-HPLC)[10,11]. The contents of OjF and
hyperoside were 51.67% and 7% in whole extracts, respectively. The
concentration used in the experiment was based on the dry weight of
the extract.
Experimental animals Beijing
ducklings within 1 d of hatching were obtained from an animal
breeding farm, Chinese Academy of Medical Sciences [SCXK- (Beijing)
2002-001].
Reagents Minimum essential
medium (MEM) was obtained from Gibco BRL (Gaithersburg, MD, USA).
Fetal bovine serum (FBS) and G-418 were purchased from HyClone
(Logan, Utah, USA). L-glutamine was obtained from Sigma (St
Louis, MO, USA). HBsAg and HBeAg enzyme immunoassay (EIA) kits were
purchased from China Isotope Co (Beijing, China).
Cell culture and treatment
HepG2.2.15 cells (clonal cells derived from human hepatoma cell line
G2) were from the Institute of Medicinal Biotechnology,
Chinese Academy of Medical Sciences. The 2.2.15 cells were cultured
in complete MEM (containing 10% FBS, 100 kU/L benzylpenicillin,
streptomycin, G-418, L-glutamine 0.03%, pH 7.0) in 75-cm2
tissue culture flasks at 37 ºC in a humidified 5% CO2.
Cytotoxic studies The 2.2.15
cells were first seeded into 96-well plates (Corning Inc, Corning,
NY, USA) at a density of 1.0〜105 cells per mL and
cultured in 200 µL complete MEM containing 10% FBS. After 24 h of
incubation, cells were washed three times with phosphate-buffered
|saline (pH 7.0) and treated with different concentrations (0.125,
0.25, 0.50, 1.00, 2.00, and 4.00 g/L) of OjF in serum-free
medium for 12 d. The medium was replaced every 4 d in MEM
supplemented with various concentrations of OjF. Untreated
cells were used as control. The cell pathological changes (CPE) were
observed by microscope. The degree of CPE was graded as: all
positive cells (-), the number of negative cells <25% (+), 25%-49%
(++), 50%-75% (+++), and >75% (++++).
Determination of HBsAg and HBeAg
The 2.2.15 cells were incubated in 24-well plates at a density
of 1.0〜105 cells per mL in 1 L MEM medium containing 10%
FBS. After 24 h, the 2.2.15 cells were treated with different
concentrations of OjF (0.125, 0.25, 0.50, and 1.00 g/L) in
serum-free medium. Cells grew in the presence of drugs for 9 d with
changes of medium every 3 d. After 6 and 9 d, supernatant was
collected and performed at -20 ＜C. The HBsAg and HBeAg in culture
medium were simultaneously measured by EIA kits on d 6 and d 9.
Experimented animals infection
and drug treatment Beijing ducklings within 1 d of hatching were
inoculated intravenously with DHBV-DNA-positive serum from Shanghai
ducks (0.2 mL/animal). Seven days after infection, ducklings were
divided into five groups: the control group (normal saline); the
positive drug group (ACV, 0.1 g，kg-1，d-1); and
the OjF 0.25, 0.50, and 1.00 g，kg-1，d-1
groups. Drugs were administered orally, bid for 10 d. Serum samples
were obtained before treatment (d 0), d 5, and d 10 during
treatment, and d 3 (d 13) after the cessation of treatment. The
serums were stored at -70 ＜C for future analysis.
Viremia analysis Viremia was
assessed throughout the treatment and follow-up period by a
semi-quantitative detection of DHBV-DNA in duck serum using a
dot-blot hybridi-zation. Fifty microliters of serum was spotted
directly onto nitrocellulose filters. After denaturation and
neutralization, the filters were hybridized with a full-length DHBV
genomic DNA probe labeled with 32P. The filters were
autoradio-graphed and the spots were counted in a scintillation
counter.
Histopathological examination of
hepatocytes On d 13, each duckling was laparotomized to obtain
the liver immediately after collecting blood from the leg vein.
Fragments of the ducklings liver were fixed in 10% formalin
solution, dehydrated with ethanol solution from 50% to 100%,
embedded in paraffin and cut into 5 µm sections, and stained using
haematoxylin-eosin dye for photomicroscopic observations.
Statistics The data were
expressed as mean＼SD, and analyzed by one-way repeated-measure ANOVA
and t-test for comparisons between groups. P<0.05 was
considered statistically significant.
Results
The cytotoxicity of OjF in
2.2.15 cells OjF-induced
cytotoxicity was observed by microscope. After 12 d of incubation
with 1.00 g/L OjF, no significant difference was found from
that of the control. However, when OjF concentration
increased, cell injury caused by OjF was observed (Table 1).
The 50% toxic concentrations (TC50) was
2.28＼0.13 g/L and the maximum nontoxic concentrations (TC0)
was 1.00 g/L.
Inhibition of HBsAg and HBeAg
production in 2.2.15 cells After 9 d of incubation, HBsAg and
HBeAg production in the culture medium were determined (Table 2).
The results showed that OjF suppressed HBsAg and HBeAg
production in the 2.2.15 cells with median effective concentration
(IC50) of about 0.56 and 0.41 g/L respectively on d 6,
0.64 and 0.30 g/L respectively on d 9 (Table 3). OjF-induced
suppression of HBsAg and HBeAg production in 2.2.15 cells was also
reflected by the inhibition rate percentage (Figure 1, 2). The
inhibitory effects appeared when cells were treated with 0.25 g/L
OjF. A substantial increase of inhibitory effects was observed
from 0.50 g/L OjF. When treated with 1.00 g/L OjF, the
inhibition rate percentage on HBsAg and HBeAg in 2.2.15 cells were
both more than 50%. The inhibition rate percentage on HBsAg was
dose-dependent, and the inhibition rate percentage on HBeAg was both
time- and dose-dependent.
Inhibitory effect of OjF
on DHBV-DNA During the course of this study, no obvious side
effects were observed in animals receiving antiviral therapy or in
control animals.
The effects of OjF and ACV,
which were used for comparison, on DHBV replication in
vivo were determined by quantification of DHBV-DNA by dot-blot
hybridization. The levels of serum viral DNA were recorded in the 5
groups before the experiment. During treatment, serum levels of DHBV-DNA
decreased in all 16 ducks treated with OjF 0.50 and 1.00 g，kg-1，d
-1 (Table 4). The mean percentage inhibition of viral DNA
levels with OjF 0.50 and 1.00 g，kg-1，d-1
was 54.3% and 64.5% respectively on the last day of treatment (Table
5). But 3 days after the cessation of treatment with ACV, the viral
replication level returned to the pretreatment baseline. In ducks
treated with OjF, the effect of DHBV-DNA inhibition lasted.
No significant decrease of serum DHBV-DNA was observed during
treatment with OjF 0.25 g，kg-1，d-1. In
the control group, serum DHBV-DNA remained unaffected during the
course of the study.
Histopathological features
Histopathological profiles of the liver from model group ducklings
revealed necrosis, steatosis, and often swelling of the hepatic
cytoplasm. The protective effect of OjF was confirmed by
histopathological examinations. Administration of OjF to the
experimental animals (1.00 g，kg-1，d-1) showed
a significant improvement of the hepatocellular architecture over
the model group, as evident from a considerable reduction in
necrosis and vacuolation (Figure 3).
Discussion
Hepatitis B virus causes acute and
chronic hepatitis, which affects nearly 360 million people worldwide[12].
Chronic infection with HBV has been associated with a high risk for
the development of primary hepatocellular carcinoma[13,14].
Effective antiviral therapy against HBV infection has not been fully
developed. Studies have been hampered by the extremely narrow host
range and limited access to experimental culture systems.
Fortunately, techniques have been developed to propagate
hepadnaviridae in tissue culture[15,16] and animal
systems[17]. The 2.2.15 cells contain all HBV particles,
and the Peking ducks allow multiplication of the HBV-like virus,
which make it possible to study the various aspects of the viral
life cycle and to examine the effectiveness of potential antiviral
drugs.
In a previous study, we have shown
that the acid-base extracts of Oj could protect hepatic
cells, decrease the content of ALT, AST, and BiliT on a liver damage
model caused by carbon tetrachloride (CCl4) in rats[18],
and inhibit DHBV[7,8]. Similar results of the inhibitory
effect of Oj extracts on HBsAg and HBeAg production in
cultured 2.2.15 cells was also observed[19]. However,
what the active part of Oj against HBV is is still unknown.
OjF was obtained through extraction and separation, which
made up more than 50% in the whole extracts. It might be an active
part against HBV. Through subsequent purification of OjF,
hyperoside, persicarin, isorhamnetin, and quercetin were obtained.
Among them, the assay of hyperoside was the highest (purity>96%) ,
which was used as the criterion to control the quality of OjF.
This study demonstrated the
inhibitory effect of OjF on HBsAg and HBeAg secretion by
human hepatoma 2.2.15 cells and on serum DHBV-DNA levels of
ducklings infected with hepatitis B virus. TC50 of OjF
was 2.28 g/L and TC0 was 1.00 g/L in 2.2.15 cells, which
suggested that the inhibitory action of OjF had no
cytotoxicity. In nontoxic concentra-tions, OjF significantly
inhibited the secretion of HBsAg and HBeAg. With OjF
concentration increasing, a dose-dependent response was observed. At
a TC0 of 1.00 g/L, the inhibition rate percentage of
OjF on HBsAg and HBeAg in 2.2.15 cells were both more than 50%,
and the inhibition rate percentage on HBsAg exceeded that of OjF
on HBeAg.
These results clearly illustrate an
inhibitory effect of OjF on HBsAg and HBeAg production in
2.2.15 cells, which provide strong evidence to evaluate the effect
of drugs against HBV in a cellular model, but it is still necessary
to verify this in an animal model. Therefore, the inhibitory effect
of OjF in the duck HBV model was investigated. Our
experiments with OjF (0.50 and 1.00 g，kg-1，d-1)
in ducklings pointed to a suppressive action on DHBV replication
in vivo. With OjF 1.00 g，kg-1，d-1,
the therapy caused a more pronounced decrease (64.5%) in viremia. It
was well known that most antivirus medicines had the inevitable
rebound effect after drug cessation. This shortcoming had limited
the therapy to those diseases infected by viruses such as HB or
AIDS. The similar phenomena appeared in the positive control drug
ACV in the present study. OjF showed therapeutic effects as
well as ACV, and no difference was observed after cessation of OjF
therapy compared to OjF-treated animals. It suggested that
OjF could maintain for a long time in treating viremia of HBV
and the effect of DHBV-DNA inhibition showed a
concentration-dependent response. Histopathological examination also
confirmed the function of OjF protecting the liver in DHBV-infected
ducklings. Xiong Q et al[19] reported that
Apocynum ventun extracts containing hyperoside and quercetin had
hepatoprotective activity, and OjF contained these two
compounds. These results demonstrated the antihepatitis B virus
effect of OjF, which were consistent with antiviral activity
of Oj[7,8], making OjF a candidate for
future evaluation in patients with HBV infection.
In order to elucidate the possible
mechanism of OjF towards DHBV-DNA on DHBV-infected ducklings,
the effect of OjF on DHBV-DNA was investigated (data not
shown). Our results indicate that OjF might inhibit the
DNA-dependent DNA polymerase reaction, which results in the
termination of replication of DHBV-DNA. This hypothesis is currently
under investigation in our laboratory.
In conclusion, OjF possessed
the significant antiviral activity in vitro and in vivo,
and it was one of the main active parts of Oj against HBV.
Elucidation of the mechanism of its antiviral activity and
identification of the active components in OjF will greatly
enhance the understanding of viral gene expressions and provide new
clues to assist in the development antiviral agents in the future.
Acknowledgement
The authors wish to thank Zhuang LI
for his excellent technical support in this study. This research was
supported by Institute of Medicinal Biotechnology, Chinese Academy
of Medical Sciences.
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