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Radix astragali is a herbal remedy widely used in
traditional Chinese medicine for the treatment of diabetes,
cardiovascular diseases (CVD), and
inflammation[1]. Astragalo-side IV
(3-0-beta-D-xylopyranosyl-6-0-beta-D-glucopyrano-
sylcycloastra-genol; Figure 1) is one of the main active
ingredients of radix astragali, which has also been reported to
have a range of pharmacological actions, including
anti-diabetic[2],
anti-hypertensive[3],
anti-inflammatory[3], myocardial
protective[4], anti-heart failure effects. These
pharmacological actions are diverse, so the purpose of the present study
was to synthetically study these effects, attempt to link them,
and then attempt to find the common underlying mechanisms.
Recent researches have indicated that obesity, insulin
resistance, hypertension, dislipidemia, and atherosclerosis
always occur together and have common pathogenesis. This
cluster of metabolic and CVD risk factors has been termed
the "metabolic syndrome", and was defined by the World
Health Organization in 1999[5]. Although the underlying
pathogenesis of metabolic syndrome is still not fully clear, a
large body of evidence now indicates that insulin resistance
may be a central abnormality, and that there is a complicated
interplay between insulin resistance, adipocytes and
endothelial dysfunction that links the abnormalities of metabolic
syndrome[6].
In the present work, we studied the pharmacological
action of astragaloside IV from the perspective of metabolic
syndrome. We investigated the effects of astragaloside IV
on (1) the process of preadipocyte differentiation into
adipocytes; (2) the glucose uptake of adipocytes that have
become insulin resistant through exposure to high glucose
levels; and (3) the peroxisome proliferator-activated
receptor-g (PPARg) gene expression of preadipocytes. We then
investigated the influence of astragaloside IV on endothelial
cell (EC) viability loss and apoptosis induced by tumor
necrosis factor-a (TNF-a). Additionally, we measured the
effect of astragaloside IV on TNF-a-induced intracellular free
Ca2+ accumulation in EC.
Materials and methods
Cell culture Preadipocytes were isolated from adipose
tissue using a method modified from that of
Rodbell[7]. The epididymal adipose tissue from male Sprague-Dawley rats
(100_150 g, Zhejiang Center of Laboratory Animals) was
removed under sterile conditions and washed in D-Hanks¡¯
solution. Minced tissue was digested with 0.1% type II
collagenase (Sigma, St Louis, MO, USA). After incubation at 37
°C
for 45 min, the digest was filtered through a
250-mm nylon mesh. The digested tissue was centrifuged at
200×g for 10 min, and mature adipocytes were removed. The pellet was
resuspended in D-Hanks¡¯ solution, filtered through a
25-mm nylon mesh, and centrifuged again. The pellet was
resuspended in Dulbecco¡¯s modified Eagle¡¯s medium (DMEM;
Gibco, Grand Island, NY, USA) containing 10% fetal calf
serum (FCS; HyClone, Logan, UT, USA). Cells were plated
in 60-mm culture dishes at a density of
1.5×105_3×105
cells/mL. The cells were then subcultured every 3 d.
Primary human umbilical vein endothelial cells (HUVEC)
were cultured using the method of Jaffe et
al[8]. Briefly, human umbilical cords were acquired aseptically from a
hospital, and HUVEC were isolated by using 0.1% type II
collagenase digestion. Cells (at a density
5×104 cells/mL) were primarily cultured in RPMI-1640 (Gibco) containing 20%
FCS in 96- or 24-well culture plates (Nunc, Roskilde, Denmark)
previously coated with 0.02% gelatin. Cells grew to
confluence in 2_3 d. Both types of cells were maintained in
a CO2 incubator with 95% air and 5%
CO2 at 37 °C.
Assessment of preadipocyte differentiation
Preadipo-cytes were plated in 24-well culture plates at a density of
5×104 cells/well. After 1 d, the cells reached maximal
conflu-ence and then were treated individually with different
concentrations of astragaloside IV (3, 10, and 30 µg/mL) (Zhejiang
Conba Pharmaceutical Co, Hangzhou, China, purity above
98%) for 3 d in 500 µL of DMEM containing insulin 1
µmol/L and 10% FCS. After the removal of insulin and astragaloside,
the cells were further cultured for 8 d, during which the
medium was renewed every 3 d. After a total of 12 d,
adipogenesis was used as an indicator of the effects of astragalo-side
on preadipocyte differentiation. Cells cultured in plates were
washed 3 times with phosphate-buffered saline (PBS), and
fixed with 10% formalin in PBS for 1 h. After being stained
with 0.1 mg/mL oil red O solution for 2 h, the cells were
washed 3 times with water, and all water was then vaporized
(32 °C for 45 min). The precipitation was dissolved by
adding 100 µL isopropanol. The absorbance at 510 nm was
measured by using a microplate reader (Bio-Rad 550, Hercules,
CA, USA)[9]. For the vehicle control, 1%
Me2SO was used (normal group), and for the positive control,
rosiglitazone 3 µg/mL (GlaxoSmithKline, Philadelphia, PA, USA) was used.
Analysis of mRNA expression by reverse
transcription_polymerase chain reaction Preadipocytes were plated on
6-well culture plates, as described in the previous paragraph.
Total RNA was isolated from adipocytes at d 7 by using a
Trizol total RNA extraction kit (Shanghai Sangon, Shanghai,
China). The reverse transcription-polymerase chain
reaction (RT-PCR) was carried out as described
previously[10].
The PCR primer sequences were as follows: aP2 forward
5¡¯-GACCTGGAAACTCGTCTCCA-3¡¯ and reverse 5¡¯-CATGA-CACATTCCACCACCA-3¡¯;
PPARg forward 5¡¯-AACCGGAA-CAAATGCCAGTA-3¡¯ and reverse
5¡¯-TGGCAGCAGTGGAA-GAATCG-3¡¯. Total RNA was reverse transcribed to cDNA
by using reverse transcriptase (Promega, Madison, WI, USA)
at 37 °C for 1 h . The cDNA was then amplified by using
Taq polymerase (Takara, Shiga, Japan) with each primer. The
temperature program for the amplification was as follows: 30
s at 94 °C, 30 s at 54 °C and 1 min at 72 °C for 23 cycles (aP2)
or 35 cycles (PPARg). The RT-PCR products were
electrophoresed on 1.4 % agarose gels, stained with ethidium
bromide, and revealed by using ultraviolet irradiation. The
RT-PCR products were semi-quantitated by using Gel Doc
2000 (Bio-Rad) and Quantity One software (Bio-Rad). The
concentration of PPARg mRNA was expressed as the ratio of
the mRNA expression of PPARg to that of b-actin.
Glucose uptake study Glucose uptake by the adipocytes
was determined by measuring the transport of
2-deoxyglu-cose (2-DG) into the cells, as described
previously[11], with some minor modifications. Preadipocytes were induced to
differentiate into adipocytes by incubation in a medium
containing 10% FCS, 1 µmol/L insulin, 1 µmol/L dexamethasone,
and 0.5 mmol/L isobutyl-methylxanthine (IBMX; Sigma) for
2 d. Then the medium was switched to one containing 10%
FCS and 1 µmol/L insulin for 2 d, and then again to a normal
10% FCS medium for 2 d. After 6 d, almost all of the
preadipocytes had differentiated into adipocytes. These cells
were plated on 24-well plates, and incubated with DMEM
medium containing a high concentration of glucose (35
mmol/L) (to induce insulin resistance) and varying concentrations
of astragaloside IV (3, 10, and 30 mg/mL), rosiglitazone (3
µg/mL) or vehicle for 48 h. Then adipocytes were
serum-deprived for 4 h, and incubated in
N-2-hydroxyethylpiperazine-N¡¯-2-ethanesulfonic acid (HEPES; Hyclone, Logan, UT, USA)
with 0.1 µmol/L insulin, 1 µCi/mL
[2-3H] deoxyglucose (Beijing Atom HighTech Co, Beijing, China) and 125 µmol/L
unlabeled 2-DG (Sigma) for 1 h. The cells were then extensively
washed with cold HEPES. After 100 µL NaOH solution (0.2
mol/L) was added, the solution was neutralized by the
addition of 100 µL HCl (0.2 mol/L). An aliquot of 150 µL of
solution was aspirated, and the radioactivity therein was
measured by liquid scintillation counting (Wallac 1414, Turku,
Finland).
Assessment of endothelial cell
apoptosis The effect of astragaloside IV on
TNF-a-induced apoptosis of EC[12] was investigated by using an annexin-V kit (Caltag, Burlingame,
CA, USA) and flow cytometer (FACsort, Becton-Dickinson,
San Jose, CA, USA). EC were plated in 24-well plates at a
density of 1×105 cells/well. After 24 h the cells were treated
individually with different concentrations of astragaloside
IV (3, 10, and 30 µg/mL), rosiglitazone (30 ng/mL) or vehicle for
48 h in 200 µL of RPMI-1640 medium containing 1 µmol/L
insulin, 10% FCS and 40 ng/mL TNF-a (Sigma). Following
culture, cells were harvested and washed twice with D-Hanks¡¯
solution, then cells were collected by centrifugation at
240×g and resuspended in 1×binding buffer at a concentration of
1×106 cells/mL. Cells in binding buffer (100 µL) were
transferred to a 5-mL culture tube, and stained with 5 µL
fluorescein isothiocyanate (FITC)-conjugated annexin V and 10 µL
propidium iodide (PI; 50 µg/mL). After 15-min incubation at
20_25 °C in the dark, 200 µL of 1×binding buffer was added
to the cells in each tube, and the mixture was analyzed on a
flow cytometer. Forward scatter (FSC) and side scatter (SSC)
were collected in linear mode and FL1 and FL2 in log mode.
At least 10 000 cells were collected for each sample, and the
data were analyzed using CellQuest software
(Becton-Dickinson). This assay identified normal cells as
PI-negative and annexin V (FITC)-negative, and apoptotic cells as
PI-negative and annexin V (FITC)-positive.
Assessment of endothelial cell viability
The effect of astragaloside IV on the TNF-a-induced viability loss of EC
was assessed by using the water-soluble tetrazolium-1
(WST-1, Dojindo Laboratories, Kumamoto, Japan) colorimetric
assay[13]. EC were plated on 96-well plates at a density of
1×104 cells/well. After 12 h, the cells were treated individually with different
concentrations of astragaloside IV (3, 10, and 30
mg/mL), rosiglitazone (30 ng/mL) or vehicle for 48 h in 100
mL of RPMI-1640 medium containing 40 ng/mL TNF-a (Sigma), 1 µmol/L
insulin and 10% FCS. After the drug treatment, WST-1
reagent was added to each well and the cells were incubated
for 2 h at 37 °C. Following incubation, absorbance at 450 nm
was determined by using a microplate reader.
Fluo-3 fluorescence measurements Fluo-3 fluorescence
was measured using a method described
elsewhere[14] with minor modifications. Briefly, cells were washed 3 times with
RPMI-1640 without FCS. Intracellular free
Ca2+ was labeled with Fluo-3 AM (Molecular Probes, Eugene, OR, USA). Cells
were incubated individually with different concentrations of
astragaloside IV (3, 10, and 30 µg/mL) or vehicle in 50
mL RPMI-1640 medium containing 5 µmol/L Fluo-3 AM at 37 °C
for 30 min. The fluorescence intensity was measured by
using laser confocal scanning microscopy (Zeiss LSM510,
Jena, Germany). The Fluo-3 AM was excited at 488 nm by
laser and emissions between 515-560 nm were obtained.
Images of 512×512 pixels in size were acquired with a 20×
objective. The acquisition rate was 1 frame (512×512 pixels)
per 5 s. After the parameters had been adjusted appropriately,
laser scanning was used to obtain a time series of images.
The TNF-a (40 ng/mL) was added after 3 s. The images
obtained were quantitatively analyzed for changes in
fluorescence intensity within regions of interests (ROI) using
the Zeiss LSM software. By selecting ROI, information about
these areas could be obtained and information such as
intensity and histograms could be further extracted. Increases
in intracellular free Ca2+ were expressed as the ratio of
fluorescence intensity of Fluo-3 AM to baseline fluorescence
intensity (F/F0).
Statistical analysis Data are presented as mean±SD.
Statistical comparisons between groups were carried out
using Student¡¯s t-test. P<0.05 was considered significant.
Results
Differentiation of preadipocytes Rosiglitazone was used
as the positive control, because it belongs to a novel class
of anti-diabetic agents, the thiazolidinediones (TZD), which
have therapeutic potential to improve metabolic syndrome
in addition to diabetes[15]. The insulin-induced increases in
lipids in preadipocytes in the groups treated with
astragalo-side IV 3, 10, and 30 µg/mL or rosiglitazone 3 µg/mL were
significantly greater than those observed in the normal group.
There was no significant difference among the groups treated
with different concentrations of astragaloside IV (Table 1).
The adipogenesis-modulating activity of astragalo-side IV
was confirmed by using a photograph (oil red O staining)
and RT-PCR analysis of adipocyte-specific aP2 gene
expression (Figure 2A, 2B). This observation suggests that
astragaloside IV can potentiate insulin-induced preadipocyte
differentiation.
Insulin-induced glucose uptake of adipocytes
Adipo-cytes incubated in medium containing a high concentration
of glucose became insulin resistant. The glucose uptake of
control group cells descended by 44%, compared to the
normal group (Table 2). The insulin-induced glucose uptake of
the group treated with astragaloside IV 30 µg/mL was
significantly higher than that of the control group and
significantly lower than that of the group treated with rosiglitazone (3
µg/mL) and the normal group. It indicates that astragaloside
IV can potentiate the uptake of glucose by adipocytes that
have become insulin resistant by exposure to high
concentrations of glucose.
PPARg expression in preadipocyte At d 7, the
concentration of PPARg mRNA transcripts in preadipocytes of the
group treated with astragaloside IV 10 µg/mL or 30 µg/mL
was significantly higher than that in the normal group. There
were no significant differences between the groups treated
with astragaloside IV 30 µg/mL or 10 µg/mL (Figure
3). This finding shows that astragaloside IV promotes
PPARg mRNA expression.
Endothelial cells dysfunction Apoptosis of endothelial
cells was significantly increased by 19.7% in the group treated
with TNF-a (40 ng/mL) relative to the normal group. The
percentage of apoptotic endothelial cells in the groups treated
with 10 and 30 µg/mL astragaloside IV and rosiglitazone (30
ng/mL) was significantly lower than that in the control group
and significantly higher than that in the normal group. There
was no significant difference between the groups treated
with 30 µg/mL and 10 µg/mL astragaloside IV, but the
percentage of apoptotic endothelial cells in the group treated
with astragaloside IV 30 mg/mL was significantly lower than
that in the group treated with 3 µg/mL astragaloside IV (Figure
4A, 4B).
Endothelial cell viability decreased by 30% relative to
the normal group after exposure to TNF-a (40 ng/mL) for 48
h. There was no significant difference with respect to cell
viability between the groups treated with rosiglitazone (30
ng/mL) and 30 µg/mL astragaloside IV, but cell viability in these
groups was significantly higher than that in the control group,
and significantly lower than that in the normal group. The
groups treated with 3 µg/mL and 10 µg/mL astragaloside IV
did not have significantly higher cell viability than the
control group. These data indicate that astragaloside IV
dose-dependently prevents endothelial cells apoptosis and
viability loss due to TNF-a.
Ca2+ elevation induced by TNF-a in
EC After being stimulated with TNF-a, the fluorescence value of
Ca2+ in EC rapidly increased and arrived at a peak value (5.6-fold) within 60
s, then slowly decreased near to the basal level (Figure 6). When
cells were pretreated with astragaloside IV, the peak
fluorescence intensity corresponding to TNF-a-induced
Ca2+
elevation was significantly reduced by 2.9-fold relative to
cells treated with TNF-a alone. It suggests that astragaloside
IV has antagonistic effects on TNF-a-induced
Ca2+ elevation in EC.
Discussion
The results of the present study showed that
astragalo-side IV could potentiate the insulin-induced differentiation
of preadipocytes. Recently, adipocytes have been
recognized to play an active role in glucose and lipid metabolism
by depositing fat and secreting polypeptides such as leptin,
resistin, and adiponectin[16]. Preadipocytes differentiate into
adipocytes, and newly differentiated lean adipocytes are
more sensitive to insulin and secrete fewer harmful
mediators (free fatty acids, TNF-a, interleukin-6,
etc) than do old obese
adipocytes[17]. Therefore preadipocyte differentiation
may play an important role in lipid and glucose metabolism.
This effect of astragaloside IV is consistent with that of
rosigli-tazone, which can also potentiate preadipocyte
differentia-tion.
Many researches indicate that insulin resistance may be
the central abnormality of metabolic
syndrome[5,6]. The presence of obese adipocyte-derived mediators (free fatty acids,
resistin, TNF-a, etc) can lead to the development of insulin
resistance. Then, through direct and/or indirect mechanisms,
insulin resistance causes hyperglycemia, endothelial
dys-function, hypertension and
arteriosclerosis[18]. In the present study, we found that similar to rosiglitazone, astragaloside
IV, at a high concentration, could potentiate the
glucose-uptake of adipocytes in which insulin resistance had been
induced by exposure to high concentrations of glucose.
These results are compatible with previously research in
which it was found that astragalus or astragalus
polysaccharides influenced carbohydrate metabolism and
preadipo-cyte differentiation[19,20].
To understand the mechanism by which astragaloside IV
potentiates preadipocyte differentiation and reduces insulin
resistance, the effect of astragaloside IV on
PPARg gene expression was investigated. PPARg is a nuclear receptor
that plays a regulatory role in the expression of genes
related to preadipocyte differentiation, insulin sensitivity, and
inflammation[21]. Rosiglitazone is thought to mainly exert its
effect through the activation of PPARg. In the present study,
we found that astragaloside IV significantly promoted
PPARg mRNA expression. Thus, astragaloside IV may, through
stimulating PPARg expression, potentiate preadipocyte
differentiation and improve insulin sensitivity. Further
investigations are needed to confirm this finding.
EC are known to play a pivotal role in vascular function
and remodeling. Endothelial dysfunction may link lipid and
glucose metabolic disorders and CVD[22]. Several
mechanisms may contribute to endothelial dysfunction in
metabolic syndrome, including insulin resistance, and the action
of inflammatory cytokines. There is reasonable evidence
showing that metabolic syndrome is an inflammatory state,
which is associated with an increase in plasma inflammatory
cytokine concentrations, particularly that of
TNF-a[23]. The inflammatory cytokines may be primarily over-released from
the obese adipocytes[24]. Accordingly, we used
TNF-a to induce inflammatory endothelial dysfunction and
investigated the effects of astragaloside IV on apoptosis and
viability loss in EC.
Apoptosis is a process for disposing of senescent,
injured, or redundant cells through self-destruction, and has
been demonstrated to play a role in EC loss during
hypertension[25]. TNF-a-induced apoptosis of EC also plays an
essential role in the pathological processes of
atherosclerosis[26]. Therefore, apoptosis represents an important process in the
pathogenesis of endothelial dysfunction. In the present
study, apoptotic cells were assessed using annexin V
labeling. We showed that astragaloside IV could reduce the
TNF-a-induced apoptosis of EC. Additionally, by using the
WST-1 assay to measure EC viability, we showed that
astragaloside IV could significantly prevent TNF-a-induced
viability loss in EC. These results are compatible with
previous findings showing that astragaloside IV can inhibit
histamine-induced inflammation in
EC[27].
Ca2+ is a major second messenger, and intracellular free
Ca2+ overload can lead to dysfunction in EC, which has been
implicated in the signaling pathways inducing
apoptosis[28]. Also, research has shown that treatment of EC with a
Ca2+ chelator (BAPTA-AM) partially prevents
TNF-a-induced apoptosis[29]. Data from the present study showed that
astragaloside IV inhibited intracellular free
Ca2+ accumulation in EC subjected to
TNF-a. These findings are compatible with the recent finding that astragaloside IV can reduce
the excessive accumulation of intracellular calcium within
myocardial cells[4]. This phenomenon may partially explain
the anti-apoptotic effect of astragaloside IV.
In conclusion, the results of the present study indicate
that astragaloside IV can potentiate preadipocyte
differen-tiation, improve insulin resistance in adipocytes exposed to
high concentrations of glucose, and prevent endothelial
apoptosis and viability loss. These effects are probably
partly due to the promotion of PPARg expression and partly
due to inhibition of abnormal EC intracellular free
Ca2+ accumulation. Thus, the present study provides new
insights into the mechanism by which astragaloside IV exerts
its effects.
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