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Introduction
Jujuboside A (JuA) is a main effective component of
jujubogenin, extracted from the seed of Ziziphus
jujuba Mill var spinosa (Bunge) Hu ex H F Chou (Ziziphus), which is
widely used in treating symptoms of insomnia and
anxiety[1,2]. However, most previously published studies have only
focused on the investigation of behavioral changes. Recent
experimental results have suggested that a high dose of JuA
could inhibit the hyperactivity of the hippocampal CA1 area
induced by penicillin sodium[2]. JuA had inhibitory effects
on the Glu-mediated excitatory signal pathway in the
hippo-campus and hippocampal formation in
vivo and in
vitro[3,4]. Those studies suggested that JuA might play the role of an
inhibitor on the central nervous system, especially on the
hippocampus. However, they could not explain the
functions of JuA on the hippocampus, and thus, the inhibitory
effect of JuA on the hippocampus is still elusive.
In the present study, we adapted the protocol of the
differential display polymerase chain reaction (DD-PCR) to
screen for differentially-expressed genes modulated by JuA
at the transcription level in the mouse hippocampus. We
found genes modulated by JuA, and explained the partial
molecular mechanism of JuA on the hippocampus at the gene
transcription level. The use of DD-PCR is universal in
investigating molecular pharmacological mechanisms of the
effective component of a Chinese traditional herb at the gene
transcription level.
Materials and methods
Chemicals and animals JuA was provided by the
National Institute for the Control of Pharmaceutical and
Biological Products in China (Beijing, China) with a purity above
98%. KM (Kunming strain) mice (male, Grade II, weighing
18_22 g) were obtained from the Laboratory Animals Center
of Sichuan Academy of Medical Sciences (Chengdu, Sichuan,
China), and maintained in an air-conditioned room with
controlled temperature (23_25 °C) and humidity (50%_70%). The
mice were housed in cages under a 12 h light/dark cycle with
access to food and water. All the animal experiments were
carried out from 19:00 PM to 23:00 PM. The animal
experiments were performed with institutional ethical approval of
protocol, and all efforts were made to minimize animal
suffering. The mice were randomly divided between the
control and the treated groups. The treated group was given an
ip injection of JuA at 80 mg/kg, and the control group was
given an equal volume of saline. Half an hour after the
injection, the mice in the control (n=10) and treated groups
(n=10) were observed for spontaneous activity. Meanwhile,
the control group (n=6) and the treated group
(n=6) were used for RNA extraction.
Spontaneous activity The mice were put in a ZZ-6
monitor (Chengdu Technology and Market Co, Ltd, Chengdu,
Sichuan, China) for observation of spontaneous activity and
adapted to the environment within 5 min. We then recorded
the number of mouse movements within a 10 min period.
After recording the number of mouse movement before any
treatment, the mice were taken out and injected with JuA or
saline and housed in the cages. Half an hour after the
injection of JuA or saline, the frequency of mouse movement was
recorded again using the same method.
RNA extraction All the animals were sacrificed by
decapitation and the hippocampi were removed immediately.
The total RNA of the hippocampus was isolated with Trizol
(Molecular Research Center Inc, Cincinnati, OH, USA)
according to the manufacturer's protocol. To remove DNA
contamination, the RNA samples were treated with RQ1
RNase-free DNase (Promega, Madison, WI, USA) and
extracted with phenol:chloroform. Finally, the RNA samples
were resuspended in 50 µL of nuclease-free water. The
concentrations of the total RNA were measured with a
biopho-tometer, and the
OD260/OD280 ratio of all RNA samples were
up to 2.0.
DD-PCR The first strand cDNA was synthesized using
the RevertAidTM First Strand cDNA Synthesis Kit (MBI
Fermentas, Burlington, Ontario, Canada). For each reaction,
4 µg of deoxyribonuclease-treated total RNA was used for
the reverse transcription, in a mixture of 2 µg 3'-anchored
primers (T15A, T15C, and
T15G, respectively) in a final volume of 12 µL, and heated at 70 °C for 5 min. Then 4 µL of 5×
reverse transcription buffer, 1 µL of 20 U/µL,
RibolockTM Ribonulease inhibitor (MBI Fermentas, Burlington, Ontario,
Canada), and 2 µL of 10 mmol/L dNTP(dATP, dCTP, dGTP,
dTTP) mix were added; the samples were incubated at 37 °C
for 5 min. Then 1 µL (200 units) of
RevertAidTM M-MuLV reverse transcriptase was added to each sample (with a final
volume of 20 µL). The mixture was incubated at 42 °C for 60
min, and finally heated at 70 °C for 10 min.
PCR was performed in 25 µL reaction mixtures containing
2 µL cDNA, 2 µmol/L 3' anchored primers and 5' arbitary
primers (Table 1), 200 µmol/L dNTP, and 2 units of
Taq DNA polymerase, with an initial denaturation at 94 °C for 3 min,
followed by 4 cycles at 94 °C for 1 min, 34 °C for 4 min, and
72 °C for 1.5 min, and 40 cycles at 94 °C for 1 min, 38 °C for
2 min, and 72 °C for 1.5 min, with a final extension at 72 °C for
10 min. After PCR amplification, 10 µL of PCR products were
separated by electrophoresis on 8% denaturing
polyacrylamide gels, and the gels were stained by
silver[5].
Re-amplification, ligation, and transformation of PCR
products and sequencing Differentially-expressed bands
were retrieved from the polyacrylamide
gels[6] and reamplified using the corresponding primer sets in 50 µL reaction
mixtures containing 5 µL recycled DNA, and 1 µmol/L primers,
respectively, 400 µmol/L dNTP and 5 units of
Taq DNA polymerase, with an initial denaturation at 94 °C for 3 min,
followed by 40 cycles at 94 °C for 1 min, 38 °C for 2 min, and
72 °C for 1.5 min, with a final extension at 72 °C for 10 min.
The reamplified PCR fragments were run on a 1% agarose
gel and gel-purified using 3S Spin Agarose Gel DNA
Purification Kit (Shenergy Biocolar, Shanghai, China) according
to the kit protocol. Products were ligated into the pUCm-T
cloning vector (Shenergy Biocolar, China) according to the
kit protocol, then transfected into Escherichia
coli high efficiency DH5α competent
cells[7] with ampicillin and blue/white selection. The positive colonies were screened by
PCR with a pair of M13 primers and digestion with restriction
endonuclease Pst I. Those colonies were sequenced by a
commercial sequencing service (Beijing Sunbiotech Co,
Beijing, China) and the DNA sequence was analyzed with
BLASTN (Bethesda, MD, USA; NCBI, National Center for
Biotechnology Information).
Semi-quantitative RT-PCR The first strand cDNA was
synthesized as protocol of DD-PCR except for 3'-anchored
primers replaced by the oligo(dT)18 primer. PCR was
performed in 25 µL reaction mixtures containing 1.5 µL cDNA, 2
µmol/L gene-specific forward and reverse primers (Table 2),
200 µmol/L dNTP, and 2 units of Taq DNA polymerase, with
initial denaturation at 94 °C for 3 min, followed by 94 °C for 1
min, 55 °C for 1 min, and 72 °C for 1 min for the number of
cycles optimized for each primer pair (Table 2) to ensure that
the product intensity fell within the linear phase of
amplification; final extension was at 72 °C for 10 min.
RT-PCR amplification of β-actin transcript was used as the
internal control to verify that equal amounts of RNA were used in
each reaction. The PCR products were run on a 1% agarose
gel, then photographed with a digital camera under UV
illumination and analyzed by Quantity One software (Bio-Rad,
Hercules, CA, USA).
Statistical analysis All data were expressed as mean±SD.
A statistical analysis of the results was carried out by
independent samples t-test and paired samples
t-test. P<0.05 was considered significant.
Results
Analysis of spontaneous activity of mice The number of
mouse movements in the control and treated groups before
any treatment was 189.4±40.4 and 187.4±45.0, respectively.
It was shown that there was no difference between the
control and treated groups before the injection of saline and JuA
(P>0.05). Half an hour after the injection of saline and JuA,
the number of mouse movements in the control and treated
groups was 178.9±47.5 and 105.7±16.8, respectively. The
analysis of independent samples t-test between the control
and treated groups after the injection of saline and JuA
suggested that JuA significantly decreased the total activity
intensity of the mice at a dosage of 80 mg/kg. The analysis
of paired samples t-test between the preinjection and
postinjection of JuA showed that JuA also decreased the
total activity intensity of the mice significantly at the same
dosage. Saline did not affect the number of mouse
movements significantly (Figure 1).
Analysis of differential expression in response to JuA
by DD-PCR and confirmed by semi-quantitative RT-PCR
To screen the differentially-expressed genes related to JuA
in the mouse hippocampus, DD-PCR was performed (Figure
2). Following the isolation and sequencing of bands
differentially-expressed between the control and treated groups
and confirmed by semi-quantitative RT-PCR (Figure 3), we
identified MAP/microtubule affinity-regulating kinase 3
(Mark3) and retinitis pigmentosa GTPase regulator
interacting protein 1 (Rpgrip1) as upregulated genes by JuA in the
mouse hippocampus.
Discussion
We found that JuA, a component extracted from a
Chinese traditional herb, significantly decreased total activity
intensity in the mice. Our results were consistent with Feng
et al[8], who confirmed that JuA had an inhibitory effect on
the spontaneous activity of mice and increased quiet state
time.
Why does JuA inhibit the spontaneous activity of mice?
Although recent studies have suggested that JuA could
inhibit the hyperactivity of the hippocampal CA1 area induced
by penicillin sodium, with inhibitory effects on the
Glu-mediated excitatory signal pathway in
hippocampus[2,3], those studies could not explain why JuA had an inhibitory effect
on the spontaneous activity of mice. In the present study,
to elucidate the inhibitory effect of JuA on mice at the gene
transcription level, we adapted the protocol of DD-PCR
invented by Liang and Pardee[9] for detecting
differentially-expressed genes in the mouse hippocampus, and the
differentially-expressed genes were confirmed by
semi-quantitative RT-PCR. Finally, we identified Mark3 and Rpgrip1 as
upregulated genes by JuA in the mouse hippocampus.
Mark3 is also known as C-TAK1 (Cdc25C-associated
kinase 1) or PAR1A[10,11], and has been implicated in cell cycle
regulation and Ras signaling through its interactions with 2
putative substrates: the Cdc25C (cell division cycle 25C)
phosphatase and the MAPK scaffold KSR1 (Kinase Suppressor of Ras
1)[10,12]. The upregulation of Mark3 mRNA by
JuA may influence cell cycle or the Ras signaling pathway
indirectly. Rpgripl, another differentially-expressed gene, is
predominantly expressed and investigated in the
retina[13_15]. It is also expressed in other regions including the heart,
spleen, and brain[16], but has been poorly investigated in
those regions. In the present study, we found that JuA
could upregulate Rpgrip1 in the mouse hippocampus, which
implies that Rpgrip1 plays an essential role in the
hippo-campus.
In conclusion, JuA evidently had inhibitory effects on
the spontaneous activity of mice; meanwhile, Mark3 and
Rpgrip1 were upregulated by JuA in the mouse hippocampus.
The relevance of JuA, the reduction of mice spontaneous
activity, and the upregulation of Mark3 and Rpgrip1 exist in
2 possible pathways. One pathway is that JuA can upregulate
Mark3 and Rpgrip1 directly or indirectly. The upregulation
of Mark3 and Rpgrip1, as well as other reasons, can inhibit
the spontaneous activity of mice. The other pathway is that
JuA can inhibit the spontaneous activity of mice through an
unknown way, and the inhibition on the spontaneous
activity of mice could induce upregulation of Mark3 and Rpgrip1.
However, although there are no studies to our knowledge
which suggest the relevance between spontaneous activity
and Mark3 or Rpgrip1, the two possible pathways suggest
that there might be an essential link between the reduction
of the spontaneous activity of mice and the upregulation of
Mark3 and Rpgrip1.
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