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Introduction
Nimodipine (NMD) is a dihydropyridine calcium channel
blocker and is currently used to prevent and treat the
ischemic damage caused by cerebral arterial spasm in
subarachnoid hemorrhage[1]. NMD has also been used in other
cerebrovascular disorders, such as ischemic
stroke[2] and multi-infarct
dementia[3].
Huanglian (Rhizoma Coptidis) and Huangqin (Radix
Scutellariae) are a familiar medicine couple in many
traditional complex prescriptions. Baicalin and berberine are the
phytochemical markers for the quality control of Radix
Scutellariae and Rhizoma Coptidis in Chinese pharmacopoeia,
respectively. Thus, we choose baicalin and berberine as the
experimental samples.
Baicalin has multiple biological activities such as
vasodilatory[4],
antioxidant[5], and antitumor
activities[6,7]. Recent studies have demonstrated that baicalin has a
protective effect against brain edema and cerebral ischemic
damage[8_10].
Berberine has varied pharmacological effects including
anti-diarrheic[11],
antimicrobial[12], and
anti-inflammatory[13]; it also exhibits protective effects against ischemic damage
after ischemia/reperfusion[14_16].
As both baicalin and berberine have beneficial effects
on brain ischemic damage, the opportunity of a combination
of them or herbs containing them with NMD is increasing. A
major concern is that a herb-drug interaction may occur.
Thus, we investigated whether baicalin and berberine
affected the transport of NMD across the blood-brain barrier
(BBB) using the primary cultured rat brain microvessel
endothelial cells (rBMEC) in the present study.
Materials and methods
Materials NMD and felodipine were provided by
Shandong Xinhua Pharmaceutical Factory (Shandong, China). Baicalin and berberine standards were purchased
from the National Institute for the Control of Pharmaceutical
and Biological Products (Beijing, China). Rhodamine123
(Rho123) was purchased from Sigma-Aldrich (St Louis, MO,
USA). Cosmic calf serum was purchased from Hyclone
(Tauranga, New Zealand). Gelatin, trypsin, and collagenase
type II were purchased from Sigma-Aldrich (USA). Dulbecco's modified Eagle's medium (DMEM, high glucose)
and Ham's F-12 nutrient mixture were purchased from Gibco
BRL (Rockville, MD, USA). Bovine serum albumin (BSA)
was purchased from SABC (Fraction V; Luoyang, He-nan,
China). Sprague-Dawley rats (7_10 d old) were supplied by
the Center of Experimental Animals, China Pharmaceutical
University (Nanjing, China). All other chemicals were of
analytical grade and commercially available.
Isolation and culture of rBMEC Primary rBMEC
isolation and culture were operated according to previous
reports[17]. Briefly, the isolated cerebral gray matter was
digested by trypsin (0.05%) at 37 °C for 20 min, then filtrated
through a 149 µm nylon mesh before the filter was collected.
The filtrate was filtered through a 79 µm nylon mesh before
the matter on the nylon mesh was collected. Then the matter
was digested by collagenase type II (0.1%) at 37 °C for 25
min, centrifuged at room temperature for 5 min
(200×g), and the cells were collected. Cells were cultured in DMEM and
F-12 supplemented with 20% cosmic calf serum under the
conditions of 37 °C with 95% air and 5%
CO2. Uptake and efflux experiments were performed in a 24-well plate when
the cells reached confluence within 12_14 d.
Uptake experiment When the cells reached confluence
within 12_14 d, uptake experiments were performed.
Cultured rBMEC were pre-incubated at 37 °C in 1 mL
Hanks' balanced salt solution (HBSS) (0.137 mol/L NaCl, 5.37
mmol/L KCl, 1.26 mmol/L CaCl2, 0.81 mmol/L
MgSO4·7H2O, 0.37 mmol/L
Na2HPO4·H2O, 0.44 mmol/L
KH2PO4, 4.17 mmol/L
NaHCO3, and 2.92 mmol/L D-glucose) for 30 min. After the
pre-incubation, the solution was removed and the HBSS (1 mL)
containing NMD (10 mg/L) or both NMD (10 mg/L) and agents
for testing was added to each incubation well. The steady
state of NMD in rBMEC was recorded at 90
min[17]. The uptake was terminated at 90 min by washing the cells 3 times
with 1 mL of ice-cold HBSS. Then the blank HBSS (0.3 mL)
was added to each incubated well, frozen, and melted
repeatedly 4 times to break down the cells. The uptake of Rho123
was performed in a similar way. After pre-incubation for 30
min with HBSS (1 mL), the solution was removed, and the
HBSS (1 mL) containing Rho123 (0.1 mg/L) or both Rho123
(0.1 mg/L) and agents for testing was added to each
incubation well. The steady state of Rho123 in rBMEC was
recorded at 120 min according to previous study in our lab. So
the uptake was terminated at 120 min by washing the cells 5
times with 1 mL of ice-cold HBSS. Then the blank HBSS
(0.5 mL) was added to each incubated well, frozen, and melted
repeatedly 4 times to break down the cells.
Efflux experiment For the efflux study, rBMEC were
incubated with NMD (10 mg/L) or Rho123 (0.1 mg/L) for 90
min, then the cells were washed 5 times with 1 mL ice-cold
HBSS. HBSS (1 mL) with or without test agents was added
to initiate the efflux at 37 °C. The efflux was terminated at the
designed time points by using the same procedure as used
in the uptake study mentioned above. Efflux was estimated
from the amount of NMD or Rho123 remaining in the cells.
Analytical method The concentration of NMD in the cell
suspension was analyzed by a HPLC system (LC-10AT,
Shimadzu, Kyoto, Japan) which was equipped with a
VP-ODS column (4.6×150 mm, 5 µm, Shimadzu, Japan) and a
UV-detector (SPD-10Avp, Shimadzu, Japan). The mobile phase
consisted of MeOH: H2O (70:30,
v/v, pH=7); the flow rate was set at 1.0 mL/min, and the analytical wavelength was 238
nm[18]. Internal standard (felodipine, 5 µg/mL, 10 µL) and 150
µL methanol were added to cell suspension (150 µL) and
mixed vigorously for 10 min, then centrifuged (44
912×g for 10 min); 200 µL of supernatant was transferred to another
clean tube and centrifuged (44 912×g for 10 min) again. After
the second centrifugalization, 120 µL of supernatant was
transferred to another clean tube and 20 µL was injected
onto the HPLC system. For the standard sample, NMD was
dissolved into a blank cell (cell without drug treatment)
suspension at a concentration from 5 to 1200 ng/mL. The lowest
limit of quantitation of NMD was 5 ng/mL in the cell
suspension. A good linearity was obtained from 5_1200 ng/mL.
The concentrations of Rho123 in the cell suspension were
determined by HPLC[19]. Twenty microliters of cell
suspension was injected into a Shimadzu
LC-10AVP system (Shimadzu, Japan) consisting of an LC-10A liquid pump, a
CTO-10ASVP column oven, and a fluorescence detector
(RF-10AXL) set at an excitation wavelength of 485 nm and
emission wavelength of 565 nm. Conditions were as follows:
column, a Shim-pack ODS (4.6 µm, 150 mm×4.6 mm id,
Shimadzu, Japan); mobile phase, 0.1% HAc (pH 4.0)_acetonitrile=3:2
(v/v); column temperature, 40 °C; flow rate, 1
mL/min. Methanol was also used to remove the protein as in
the case of NMD. For the standard sample, Rho123 was
dissolved into the blank cell (cell without drug treatment)
suspension at a concentration from 0.5 to 50 ng/mL. The
lowest limit of quantitation of Rho123 was 0.5 ng/mL in the
cell suspension. The linear range of Rho123 was 0.5_50 ng/mL.
The protein content in the cultured cells was measured
by the method of Brandford MM[20] using BSA as the
standard. Net uptake, expressed as the concentration ratio
(ng/µg protein), was obtained by dividing the apparent
uptake amount of NMD or Rho123 (ng/mL) by protein
content (µg protein/mL). All data were expressed as mean±SD.
Statistical analysis was performed by using Student's
t-test. A difference of P<0.05 was considered statistically
significant.
Results
Effects of tested agents on the rBMEC cytoactivity
In order to investigate whether tested agents affected cell
cytoactivity, cells were incubated at 37 °C for 90 min with 10
mg/L NMD, 120 min with HBSS, 0.1 mg/L Rho123, 20 µg/mL
baicalin, and 10 µg/mL berberine, respectively. Cell activity
was measured using the 3-(4,
5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The HBSS
group was set as the control group. Data were shown in
Table 1. No differences (P>0.05) were found relative to the
control group. Thus, we believed that the tested agents did
not damage the cells at the concentrations used.
Effects of baicalin on the steady-state uptake of NMD
The concentration-dependent, bidirectional effect of baicalin
on the steady-state uptake of NMD was observed. Baicalin
at 2, 5, and 10 µg/mL increased the steady-state uptake of
NMD, whereas baicalin at 15 and 20 µg/mL decreased the
uptake (Figure 1).
Effects of berberine on the steady-state uptake of NMD
Similar to baicalin, berberine also affected the uptake of NMD
in a dose-dependent, biphase manner. Figure 2 shows the
profiles of the steady-state uptake of NMD by rBMEC in the
presence or absence of berberine. Berberine increased the
uptake of NMD by primary cultured rBMEC in a dose-
dependent manner from 0.01 to 1 µg/mL, but decreased the
uptake in a dose range from 2 to 10 µg/mL. The steady-state
uptake of NMD was increased to 109%, 151%, and 202% of
the control group in the presence of 0.01, 0.1, and 1 µg/mL
berberine, respectively. The uptake of NMD decreased to
72%, 53%, and 46% of the control group by 2, 5, 10 µg/mL
berberine, respectively.
Effects of baicalin and berberine on the steady-state
uptake of Rho123 The transport of NMD across rBMEC
was reported to be restricted by P-glycoprotein
(P-gp)[17]. To clarify whether the observed effects of baicalin and
berberine on the uptake of NMD were via P-gp modulation, we
used Rho123, a typical P-gp substrate, as the positive control.
The results were illustrated in Figure 3. The steady-state
uptake of Rho123 was 106%, 123%, 108%, 86%, and 59% of
the control group in the presence of 2, 5, 10, 15, and 20 µg/mL
baicalin, respectively (Figure 3A). This result was similar to
the observations in the uptake of NMD. Similar to the effect
on the uptake of NMD, berberine exhibited a
dose-depen-dent, bidirectional effect on the steady-state uptake of
Rho123 by rBMEC. The steady-state uptake of Rho123 by
primary cultured rBMEC was increased by berberine (0.01_1
µg/mL), but decreased by berberine (2_10 µg/mL; Figure 3B).
Effects of baicalin and berberine on the efflux of NMD
To further understand the mechanism of the effect baicalin
and berberine showed on the transport of NMD, we
examined the effects of baicalin and berberine on the efflux
of NMD from rBMEC. In the presence of baicalin (5 µg/mL),
the amount of NMD remaining in the cells was significantly
higher than the control group. When the cells were
incubated with 20 µg/mL baicalin, the amount of NMD remaining
in the cells significantly decreased (Figure 4). These results
suggested that 5 µg/mL baicalin inhibited the efflux of NMD,
while 20 µg/mL baicalin stimulated the efflux of NMD from
rBMEC. Similarly, the resident amount of NMD in the cells
was significantly increased by berberine (1 µg/mL), but
decreased with berberine (10 µg/mL; Figure 4), which
indicated that the efflux of NMD from rBMEC was inhibited by 1
µg/mL berberine, but promoted by 10 µg/mL berberine.
Effects of baicalin and berberine on the efflux of Rho123
As in the uptake experiment, the effects of baicalin and
berberine on the efflux of Rho123 were also examined to provide
a positive control. Similar with the effect on the efflux of
NMD, 5 µg/mL baicalin and 1 µg/mL berberine inhibited the
efflux of Rho123, while 20 µg/mL baicalin and 10 µg/mL
berberine stimulated the efflux (data were shown in Figure
5).
Discussion
The present study demonstrated that both baicalin and
berberine affected the transport of NMD across the BBB in a
bidirectional manner.
The MTT assay demonstrated that tested agents did not
damage cells at the concentrations used; we believed that
the observed changes were not the result of nonspecific
cytotoxicity.
We observed a concentration-dependent, bidirectional
effect of baicalin on the steady-state uptake of NMD by
primary cultured rBMEC; the uptake was significantly
increased by 5 µg/mL baicalin, but decreased with 20 µg/mL
baicalin. In the efflux experiment, baicalin (5 µg/mL)
inhibited the efflux of NMD while baicalin (20 µg/mL) stimulated
the efflux. Baicalin belongs to the class of flavonoids and in
recent years, flavonoids had been described as P-gp
modu-lators, however, contradictory effects have been reported.
Critchfield et al[21] reported that quercetin increased
adria-mycin efflux from HCT-15 colon cells, whereas Scambia
et al[22] found that quercetin inhibited Rho123 efflux in MCF-7
breast cells. Gwenaelle et
al[23] demonstrated flavonoids as a new class of bifunctional modulators of P-gp and Yoshiharu
et al[24] reported a concentration-dependent biphasic effect
of quercetin on the uptake of [3H]vincristine, which was a
similar phenomenon to our result. The effect of baicalin on
the uptake and efflux of Rho123, the positive control, has the
same tendency as the effect on NMD uptake; baicalin (5
µg/mL) inhibited the efflux and increased the uptake of Rho123,
whereas 20 µg/mL baicalin stimulated the efflux and
significantly decreased the uptake of Rho123. These results
suggested that the effect of baicalin on the transport of NMD
across the BBB might due to the modulation of P-gp function.
Berberine also showed a dose-dependent bidirectional
effect on the transport of NMD across the BBB. Berberine
increased the uptake of NMD from 0.01 to 1 µg/mL and
decreased the uptake from 2 to 10 µg/mL. The efflux of NMD
was inhibited by 1 µg/mL berberine and was promoted by 10
µg/mL berberine. We also adopted Rho123 as a positive
control. Berberine demonstrated the similar effect on the
transport of Rho123 as on the transport of NMD. Our
results suggested that the effect of berberine on the
transport of NMD across the BBB might result from the
modulation of berberine on P-gp function. Sun et
al[25] reported that berberine (30 µmol/L, corresponding to 10 µg/mL) decreased
the uptake of carbamazepine by rBMEC, which was in
agreement with our result. Verapamil had been reported to
modulate P-gp ATPase activity in a bidirectional
manner[26]. Whether berberine affected P-gp function via the same path
still needs further investigation.
As the concentrations of baicalin (<10
µg/mL)[27] and berberine (<100 ng/mL
level)[28] in vivo were so low, they
might only be able to demonstrate inhibitory effects on P-gp
function in in vivo studies. In addition, the detail
mechanism of the bidirectional effect observed in the present study
still needs further investigation.
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