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Life Sciences 81 (2007) 509 – 518
www.elsevier.com/locate/lifescie
Protective effects of ginsenoside Rb1, ginsenoside Rg1,
and notoginsenoside R1 on lipopolysaccharide-induced
microcirculatory disturbance in rat mesentery
Kai Sun a , Chuan-She Wang a,c , Jun Guo a , Yoshinori Horie b , Su-Ping Fang a , Fang Wang a ,
Yu-Ying Liu a , Lian-Yi Liu a , Ji-Ying Yang a , Jing-Yu Fan a , Jing-Yan Han a,b,c,⁎
a
c
Tasly Microcirculation Research Center, Peking University Health Science Center, Beijing, China
b
Department of Internal Medicine, School of Medicine, Keio University, Tokyo, Japan
Department of Integration of Chinese and Western Medicine, School of Basic Medical Sciences, Peking University, Beijing, China
Received 18 January 2007; accepted 19 June 2007
Abstract
Ginsenoside Rb1 (Rb1), ginsenoside Rg1 (Rg1), and notoginsenoside R1 (R1) are major active components of Panax notoginseng, a Chinese
herb that is widely used in traditional Chinese medicine to enhance blood circulation and dissipate blood stasis. To evaluate the effect of these
saponins on microcirculatory disturbance induced by lipopolysaccharide (LPS), vascular hemodynamics in rat mesentery was observed
continuously during their administration using an inverted microscope and a high speed video camera system. LPS administration decreased red
blood cell velocity but Rb1, Rg1, and R1 attenuated this effect. LPS administration caused leukocyte adhesion to the venular wall, mast cell
degranulation, and the release of cytokines. Rb1, Rg1, and R1 reduced the number of adherent leukocytes, and inhibited mast cell degranulation
and cytokine elevation. In vitro experiments using flow cytometry further demonstrated that a) the LPS-enhanced expression of CD11b/CD18 by
neutrophils was significantly depressed by Rb1 and R1, and b) hydrogen peroxide (H2O2) release from neutrophils in response to LPS stimulation
was inhibited by treatment with Rg1 and R1. These results suggest that the protective effect of Rb1 and R1 against leukocyte adhesion elicited by
LPS may be associated with their suppressive action on the expression of CD11b/CD18 by neutrophils. The protective effect against mast cell
degranulation by Rb1 and R1, and the blunting of H2O2 release from neutrophils by Rg1 and R1 suggest mechanistic diversity in the effects of
Panax notoginseng saponins in the attenuation of microcirculatory disturbance induced by LPS.
© 2007 Published by Elsevier Inc.
Keywords: Panax notoginseng; Microcirculatory disturbance; Leukocyte adhesion molecules; Hydrogen peroxide; Mast cell degranulation; Lipopolysaccharide
Introduction
Lipopolysaccharide (LPS) is a component of the outer cell wall
of Gram-negative bacteria, which gives rise to various manifestations of Gram-negative sepsis and septic shock (Opal et al., 1999).
In sepsis and shock, activation of inflammatory cells and the
⁎ Corresponding author. Chairman of Department of Integration of Chinese
and Western Medicine, School of Basic Medical Sciences, Peking University,
No. 38 Xueyuan Road Haidian District Beijing 100083, China. Tel.: +86 10
8280 2862; fax: +86 10 8280 2996.
E-mail address: [email protected] (J.-Y. Han).
0024-3205/$ - see front matter © 2007 Published by Elsevier Inc.
doi:10.1016/j.lfs.2007.06.008
excessive production of proinflammatory mediators lead to tissue
injury, multiple organ failure, and death (McCuskey et al., 1996).
Leukocyte recruitment in blood vessels has been documented
to be a crucial step in this process (Woodman et al., 2000). LPS
has been shown to upregulate CD11b/CD18 in neutrophils
(Liu et al., 2005), which enables leukocytes to bind to the vessel through interaction with intercellular adhesion molecules-1
(ICAM-1, CD54) on the surface of the endothelium (Carlos and
Harlan, 1994). These leukocyte–endothelial interactions promote
the release of reactive oxygen species (ROS) and other mediators,
which, on one hand, destroy bacteria, but at the same time inflict
damage on the endothelium and cause microvascular dysfunction
(Granger and Kubes, 1994).
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K. Sun et al. / Life Sciences 81 (2007) 509–518
The current management of sepsis includes early clinical
optimization of hemodynamic status together with other physiologically supportive measures, timely use of antimicrobial agents,
and elimination of the source of infection (Raghavan and Marik,
2006). A great number of recent advances in cellular and molecular biology have resulted in the development of various novel
molecular approaches to severe sepsis. These approaches target a
variety of interdependent mediators. The sites of action range from
the initiators (i.e. LPS, tissue factors) to various molecular mediators (i.e. tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1),
platelet activating factor (PAF)) as well as neutrophil receptors
(Otero et al., 2006). Despite encouraging results in animal studies,
human clinical results with these newer agents investigated in the
early 1990s targeting various aspects of the inflammatory response
were largely disappointing, demonstrating little impact on 28-day
mortality after the start of therapy (Dhainaut et al., 2001). It is clear
that the physiological disturbance in the microcirculation during
sepsis is a complicated process.
The herbal formulation Panax notoginseng (PN) is derived
from the root of the traditional Chinese herb P. notoginseng,
which is widely used in China to improve blood circulation and
ameliorate pathological hemostasis (Sun et al., 2003; White et al.,
2000). P. notoginseng saponins (PNS), the main active components of PN, include more than 30 different types of saponins (Du
et al., 2003), among which ginsenoside Rb1 (Rb1), ginsenoside
Rg1 (Rg1), ginsenoside Rd (Rd), and notoginsenoside R1 (R1)
are found in the highest content (Li et al., 2005a,b). A number of
clinical and physiological effects of PNS have recently been
described, such as improvement of myocardial dysfunction in rats
with burn injuries (Zhang et al., 2003), amelioration of hepatic
microcirculatory disturbances (Park et al., 2005), attenuation of
LPS-induced adhesion of leukocytes to the venular wall and
suppression of CD11b/CD18 expression in rat neutrophils (Sun
et al., 2006), and improvement of endothelial cell function (Chen
et al., 2004). Cardiotonic pills (CP, a traditional Chinese medicine
which contains PN, salvia miltiorrhiza, and borneol), which use
PNS as major ingredients, attenuate adhesion of leukocytes to
hepatic sinusoids after ischemia–reperfusion (I/R) in rats chronically fed with ethanol, as demonstrated in our previous studies
(Horie et al., 2005). In general, it can be said that the cellular and
molecular events which underlie the biological activity of the
major components of PNS have attracted much attention in the
past few years. In another work, Rg1 was revealed to have a
potent anti-oxidative effect and to be able to increase the activity
of superoxidase (SOD) in the blood (Chen et al., 2003). It was also
reported that R1 could depress LPS-induced expression of TNF-α
by endothelial cells in vitro, and this effect was related to its
inhibition of the degradation of inhibitory factor-κBα (I-κBα)
(Zhang et al., 1997). An inhibitory effect on LPS-induced expression of TNF-α, as well as interleukin-6 (IL-6), was also
reported for Rb1 (Smolinski and Pestka, 2003). However, no
previous investigation has specifically evaluated the roles of each
individual component of PNS, particularly the principal saponins
Rb1, Rg1, and R1, in affecting the dynamic process of microcirculatory disturbance induced by LPS.
In the present study, we evaluated changes in venular diameter, red blood cell (RBC) velocity, and leukocyte adhesion to
and emigration across the venular wall, together with mast cell
degranulation in the mesentery of rats after LPS administration,
and investigated the effects of Rb1, Rg1, and R1 on LPS-induced
microcirculatory disturbance.
Materials and methods
Reagents and animals
Rb1 and Rg1 were purchased from the National Institute for
the Control of Pharmaceutical and Biological Products. R1 was
purchased from the Kun Ming Feng-Shan-Jian Medical Company. All of these saponins were chromatographically pure, and
their structures are shown in Fig. 1.
Male Sprague–Dawley (SD) rats weighing 200–250 g were
obtained from the Animal Center of Peking University Health
Science Center. The certificate code of these animals was SCXK
2002-0001. The rats were fed a standard laboratory chow diet
(Animal Center of All Animals Peking University Health
Science Center) for 72 h before the experiment and maintained at
24 ± 1 °C and a relative humidity of 50 ± 1% with a 12/12-hour
light/dark cycle. The animals were fasted for 12 h before the
experiment, but allowed free access to water. All animals were
handled according to the guidelines of the Peking University
Animal Research Committee.
Microcirculation in the mesentery
Rats were anesthetized with urethane (1.25 mg/kg body
weight) by intramuscular injection (i.m.), and the abdomen of
each rat was opened via a midline incision (20–30 mm in
length). The jugular vein and femoral vein were cannulated for
injection of various reagents. The ileocecal portion of the
mesentery (10–15 cm region of caudal mesentery) was gently
drawn out, exteriorized and mounted on a transparent plastic
stage. The mesentery was kept warm and moist by continuous
superfusion with saline solution at 37 °C. Microcirculatory
hemodynamics in the mesentery was observed by a transillumination technique using an inverted microscope (DM-IRB,
Leica, Germany). A video camera (Jk-TU53H, Toshiba, Japan)
mounted on the microscope transmitted images to a color
monitor (J2118A, TCL, China), and the images were recorded
onto a DVD (DVR-R25, Malata, China). Single unbranched
venules without an obvious bend and with diameters ranging
between 30 and 50 μm and lengths of about 200 μm were
selected for observation in this study (Han et al., 2001).
Drug administration
The rats were divided randomly into eight groups, six animals
in each group. For the control group, LPS group, and groups
receiving saponins alone, the animals were initially observed for
10 min, then given a continuous infusion via the right jugular
vein for a period of 60 min. The infusion contained saline (6 ml/
kg/h, control group), LPS dissolved in saline (2 mg/kg/h, LPS
group) (Miura et al., 1996), or with Rb1, Rg1, or R1 dissolved in
saline (5 mg/kg/h, Rb1, Rg1, or R1 alone group) (Sun et al.,
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K. Sun et al. / Life Sciences 81 (2007) 509–518
511
Fig. 1. Structures of ginsenoside Rb1 (Rb1), ginsenoside Rg1 (Rg1), and notoginsenoside R1 (R1).
25 frames/s. RBC velocity in the venule was measured with
Image-Pro Plus 5.0 software before LPS infusion (0 min), and at
20, 40, and 60 min after LPS infusion (Han et al., 2001).
2006). In the saponin plus LPS groups, after 10 min preliminary
observation, Rb1 (Rb1 + LPS group), Rg1 (Rg1 + LPS group), or
R1 (R1 + LPS group) was infused 20 min before LPS infusion
via the right jugular vein. LPS was then infused at a dose rate of
2 mg/kg/h continuously for 60 min until the end of the observation while the saponins were infused simultaneously, with
observation starting only after LPS infusion. The saponins used
in each of the three groups were dissolved in saline and given at a
dose rate of 5 mg/kg/h (Sun et al., 2006). The microcirculatory
hemodynamics in rat mesentery was examined and recorded
continuously.
Venular wall shear rate (SR) was estimated based on the
formula SR = 2.12 × 8 × [V/D], where D is the mean diameter of
the vessel, V is the mean RBC velocity and 2.12 is an empirical
correction factor obtained from velocity profiles measured in
microvessels in vivo (Dunne et al., 2002).
Determination of venular diameter
Determination of leukocyte adhesion
Venular diameter was measured from recorded video images
before LPS infusion (0 min), and at 20, 40, and 60 min after LPS
infusion, using Image-Pro Plus 5.0 software (Media Cybernetic,
USA). The diameter was taken as the mean of three measurements at one location (Han et al., 2001).
To evaluate leukocyte adhesion in venules, the dynamic behavior of leukocytes was reviewed by replaying the recorded video
images. In the present analysis, adherent leukocytes were defined
as cells that attached to the same site for more than 10 s. Before
LPS infusion (0 min), and at 20, 40, and 60 min after LPS infusion,
the number of adherent leukocytes was counted along venule
segments (30–50 μm in diameter, 200 μm in length) which were
selected randomly from the recorded images (Han et al., 2001).
Measurement of RBC velocity
The velocity of RBCs in the venule was recorded for 10 s at a
rate of 2000 frames/s by changing the monitor from a charge
coupled device (CCD) to a high speed video camera system
(FASTCAM-ultima APX, Photron, Japan), and the recordings
were replayed from the stored high speed images at a rate of
Shear rate measurement
Determination of leukocyte emigration
The number of emigrated leukocytes was estimated by reviewing the images of leukocytes recorded before LPS infusion
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Table 1
The effects of Rb1, Rg1 and R1 on RBC velocity in mesenteric venules of rats
infused with LPS
Groups
Time after LPS infusion (min)
0
20
40
60
Control
LPS
Rb1
Rg1
R1
LPS + Rb1
LPS + Rg1
LPS + R1
1.92 ± 0.43
1.90 ± 0.14
1.85 ± 0.41
1.87 ± 0.29
1.91 ± 0.42
1.87 ± 0.28
1.83 ± 0.28
1.92 ± 0.26
2.01 ± 0.27
1.52 ± 0.12
1.82 ± 0.41
1.88 ± 0.29
1.93 ± 0.46
1.81 ± 0.26
1.77 ± 0.30
1.69 ± 0.20
1.93 ± 0.17
1.41 ± 0.09 ⁎#
1.86 ± 0.41
1.83 ± 0.27
1.95 ± 0.45
1.66 ± 0.31
1.66 ± 0.24
1.88 ± 0.26
2.02 ± 0.27
1.30 ± 0.11 ⁎#
1.85 ± 0.37
1.85 ± 0.28
1.95 ± 0.48
1.79 ± 0.29
1.52 ± 0.21
1.72 ± 0.18
Time course of changes in RBC velocity in mesenteric venules. The dosage of
LPS was 2 mg/kg/h and the dosage of Rb1, Rg1, and R1 was 5 mg/kg/h. There
were 6 rats in each group. Data are expressed as mean ± S.E. ⁎ P b 0.05 vs. time
zero value, # P b 0.05 vs. control group. LPS treatment resulted in a significant
decrease in RBC velocity in rat mesenteric venules compared to the time zero
value and the control group and this decrease was significant after 40 min of
infusion. This LPS-induced decrease was ameliorated by addition of Rb1,
Rg1, or R1. Administration of Rb1, Rg1, or R1 alone had no effect on RBC
velocity.
(0 min), and at 20, 40, and 60 min after LPS infusion. Leukocyte
emigration was expressed as the number of cells per 200 μm
length of vessel, observed in the tissue surrounding the venule
(30–50 μm in diameter) randomly chosen from the images
(Kurose et al., 1997).
Determination of mast cell degranulation
Sixty minutes after LPS infusion, the tissue was infused
with 0.1% toluidine blue for 1 min and rinsed with saline.
Degranulated mesenteric mast cells were identified by the presence of intracellular granules released into the surrounding
tissue, and these cells were counted within the circular microscopic field of a 20× objective lens. Five fields surrounding the
microvasculature were evaluated for each mesenteric window.
The numbers of both nondegranulated and degranulated mast
cells were scored, and the percentage of degranulated mast cells
was calculated (Kurose et al., 1997).
Determination of plasma cytokines
After observation of mesentery microcirculation, blood
was collected from the abdominal aorta of each animal and anticoagulated with heparin (20 unit/ml blood). The plasma
was isolated by centrifugation. Fifty microliters of plasma or
standard was incubated with 50 μl capture beads for 1 h at room
temperature, and then mixed with 50 μl phycoerythrobilin
(PE)-labeled TNF-α and IL-6 detection antibodies and incubated for 2 h at room temperature to form a sandwich
complex. Following incubations, 1 ml of washing buffer (BD
Biosciences Pharmingen, USA) was added to each tube,
and the mean fluorescence intensity was detected using flow
cytometry (FACSCalibur, B.D. Co, USA). The data were analyzed with BD Cytometric Bead Array analysis software
(Wong et al., 2004).
In vitro determination of CD11b/CD18 expression
The expression of CD11b/CD18 was examined in vitro
in another group of SD rats (n = 6). The rats were fasted for 12 h
and given water ad libitum before the experiment. They were
anesthetized with urethane (1.25 mg/kg body weight, i.m.).
Blood was collected from the abdominal aorta of each animal,
anti-coagulated with heparin (20 unit/ml blood), and twenty
samples were prepared as follows: control, Rb1 (0.2 mg/ml),
Rg1 (0.2 mg/ml), R1 (0.2 mg/ml), Rb1 (1.0 mg/ml), Rg1
(1.0 mg/ml), R1 (1.0 mg/ml), Rb1 (2.0 mg/ml), Rg1 (2.0 mg/ml),
R1 (2.0 mg/ml), LPS, LPS + Rb1 (0.2 mg/ml), LPS + Rg1
(0.2 mg/ml), LPS + R1 (0.2 mg/ml), LPS + Rb1 (1.0 mg/ml),
LPS + Rg1 (1.0 mg/ml), LPS + R1 (1.0 mg/ml), LPS + Rb1
(2.0 mg/ml), LPS + Rg1 (2.0 mg/ml), and LPS + R1 (2.0 mg/ml).
Into each of these preparations, 0.2 ml of blood was added. The
control sample was 8 μl of saline. The saponin-alone samples
consisted of 4 μl saline and 4 μl of one of the three saponins, the
final concentration of which is indicated in parentheses above.
The LPS sample consisted of 4 μl saline and 4 μl LPS for a final
concentration of LPS of 2 μg/ml. All other experimental samples
consisted of 4 μl LPS and 4 μl of one of the three experimental
saponins for a final concentration of LPS of 2 μg/ml, and final
concentrations of each experimental saponin as indicated above in
parentheses. The concentrations of the drugs used in this in vitro
experiment were chosen according to our previous work (Sun
et al., 2006), in which preliminary experiments were performed to
determine the adequate concentrations for the drugs. We did not
attempt to deduce the drug concentrations used for in vitro
experiment directly from those used for in vivo experiments,
because no comparability is expected between the two systems in
terms of the issues addressed in the present study. The samples
were mixed and maintained for 2 h at 37 °C, and then incubated
with 1 μg FITC-labeled antibody against CD11b or CD18 (BD
Biosciences Pharmingen, USA) for 20 min at room temperature.
The cells were lysed with hemolysin (BD Biosciences Immunocytometer Systems, USA) and washed twice with PBS. Flow
cytometry (FACSCalibur, B.D. Co, USA) was used to assess
the mean fluorescence intensity. Neutrophils were selected by
FSC-SSC scattergram. Five thousand neutrophils were evaluated
for each sample, and the mean fluorescence intensities were
calculated.
Analysis of intracellular H2O2 and ·O2− production by
neutrophils in vitro
Blood was taken from abdominal aorta and anti-coagulated
with heparin. Neutrophils were isolated using mono-poly resolving medium, as reported by Ting and Morris (1971), and
suspended in RPMI 1640 medium for analysis of intracellular
production of H2O2 and superoxide anion (·O2-) by flow cytometry (FACSCalibur, B.D. Co, USA), according to the modified method of Shen et al. (1998). Briefly, the neutrophils were
incubated at 37 °C for 5 min with 0.2 mM 2′,7′-dichlorofluorescein diacetate (DCFH-DA) and then incubated for an additional 15 min with 0.1 mM of hydroethidine (HE). The cells were
then treated with Rb1 (1.0 mg/ml), Rg1 (1.0 mg/ml), or R1
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K. Sun et al. / Life Sciences 81 (2007) 509–518
513
Table 2
The effects of Rb1, Rg1 and R1 on venular shear rate in mesentery of rats infused with LPS
Groups
Control
LPS
Rb1
Rg1
R1
LPS + Rb1
LPS + Rg1
LPS + R1
Time after LPS infusion (min)
0
20
40
60
775.86 ± 146.74
810.17 ± 59.18
779.36 ± 121.09
792.61 ± 83.11
818.55 ± 147.98
827.43 ± 122.79
826.19 ± 122.86
863.32 ± 97.86
799.70 ± 78.22
644.05 ± 42.01
765.52 ± 126.29
755.69 ± 77.40
811.81 ± 182.51
792.63 ± 109.97
807.37 ± 135.20
747.11 ± 64.42
786.61 ± 61.86
599.80 ± 44.44 ⁎#
814.61 ± 151.28
754.18 ± 81.33
826.77 ± 186.14
726.70 ± 135.52
749.64 ± 102.64
801.64 ± 93.16
803.73 ± 98.14
553.54 ± 54.31 ⁎#
819.44 ± 140.88
747.51 ± 84.42
816.27 ± 174.18
772.75 ± 115.70
682.67 ± 91.36
742.35 ± 77.05
Changes in rat mesenteric venular shear rate over time. The dosage of LPS was 2 mg/kg/h and the dosage of Rb1, Rg1, and R1 was 5 mg/kg/h. There were 6 rats in each
group. Data are expressed as mean ± S.E. ⁎ P b 0.05 vs. time zero value, # P b 0.05 vs. control group. LPS treatment elicited a decrease in the mesenteric venular shear
rate compared to the time zero value and the control group and this decrease became significant after 40 min of infusion. Addition of Rb1, Rg1, or R1 blunted the LPSinduced decrease in shear rate. Administration of Rb1, Rg1, or R1 alone had no influence on the shear rate.
(1.0 mg/ml) alone or stimulated with LPS (2 μg/ml) simultaneously. After a 20 min incubation on ice, the production of
H2O2 and ·O2– was determined with flow cytometry by measuring emission at 525 nm for 2′,7′-dichlorofluorescein (DCF)
and 590 nm for ethidium bromide (EB).
Probability values of less than 0.05 were considered to be statistically significant.
Results
Statistical analysis was performed using ANOVA and the
F-test. Values are presented as mean ± standard error (n = 6).
No significant difference in the diameter of mesenteric
venules after LPS infusion was detected among the groups of
rats at time zero or at any time point in the entire observation
(data not shown).
The time course of changes in RBC velocity in mesenteric
venules in various conditions is depicted in Table 1. As expected, the velocity of RBCs in venules of the control group
remained unchanged over the 60 min period of observation. By
contrast, the velocity of RBCs in mesenteric venules of rats of
the LPS group progressively declined with LPS infusion, being
significantly lower at both 40 and 60 min compared to the time
zero value or the control (P b 0.05). After 60 min of infusion,
RBC velocity was measured as 1.30 ± 0.11 mm/s which was
70% of the time zero value. Treatment with Rb1, Rg1, or R1
ameliorated the LPS-induced diminution in RBC velocity in
Fig. 2. Changes in the number of leukocytes adherent to the mesenteric venules
over time. The abscissa represents time after LPS infusion. The dosage of LPS
was 2 mg/kg/h and the dosage of Rb1, Rg1, and R1 was 5 mg/kg/h. There were 6
rats in each group. Data are expressed as mean ± S.E. ⁎ P b 0.05 vs. control
group, # P b 0.05 vs. LPS group. The number of adherent leukocytes increased
progressively and linearly with LPS treatment, and this increase was attenuated
by addition of R1, Rb1, or Rg1, with significant attenuation starting at 20, 40
and 60, min, respectively. Rb1, Rg1, or R1 treatment alone had no effect on
leukocyte adhesion.
Fig. 3. Changes in the number of emigrated leukocytes outside the mesenteric
venules over time. The abscissa represents the time after LPS infusion. The
dosage of LPS was 2 mg/kg/h and the dosage of Rb1, Rg1, and R1 was 5 mg/kg/
h. There were 6 rats in each group. Data are expressed as mean ± S.E. ⁎ P b 0.05
vs. control group, # P b 0.05 vs. LPS group. The number of emigrated leukocytes
increased markedly after 40 min of LPS infusion and continued to increase to the
end of observation. R1 inhibited the LPS-induced increase in the number of
emigrated leukocytes at 60 min after LPS stimulation, while Rb1 and Rg1
had no effect. Rb1, Rg1, or R1 treatment alone had no effect on leukocyte
emigration.
Determination of plasma alkaline phosphatase
After observation of mesentery microcirculation, blood was
collected from the abdominal aorta of each animal and anticoagulated with heparin (20 unit/ml blood). The plasma was
isolated by centrifugation. Alkaline phosphatase (AP) in plasma
was determined with an AP detection kit (Olympus, US), using
an Automatic Biochemical Analyzer (7170A, Hitachi, Japan)
(Tietz et al., 1983).
Statistics
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K. Sun et al. / Life Sciences 81 (2007) 509–518
Fig. 4. Effects of Rb1, Rg1, and R1 on mast cell degranulation induced by LPS infusion for 60 min. The dosage of LPS was 2 mg/kg/h and the dosage of Rb1, Rg1, and
R1 was 5 mg/kg/h. There were 6 rats in each group. Data are expressed as mean ± S.E. ⁎ P b 0.05 vs. control group, # P b 0.05 vs. LPS group. The number of
degranulated mast cells increased markedly in response to LPS compared to the control group. Rb1 and R1 inhibited this increase, while Rg1 had no effect. Rb1, Rg1,
or R1 treatment alone had no effect on mast cell degranulation.
rat mesenteric venules to some extent. But there was no statistically significant difference detected for any of the experimental groups at any time point in comparison with either the
control group or the LPS group. Likewise, administration
of Rb1, Rg1, or R1 alone had no effect on the RBC velocity
compared to control.
There was no significant difference in the time zero value for
shear rates among the groups (Table 2). LPS infusion led to a
progressive decrease in the mesenteric venular shear rate, and a
significant difference was observed 40 min and 60 min after the
start of LPS infusion compared to the time zero value and the
control group (P b 0.05). Notably, treatment with Rb1, Rg1, or
R1 was found to ameliorate the LPS-induced reduction in the
shear rate to some extent. By contrast, administration of Rb1,
Rg1, or R1 alone had no influence on the shear rate.
Fig. 2 shows the time course of leukocyte adhesion in rat
mesenteric venules in control, LPS, Rb1 + LPS, Rg1 + LPS, and
R1 + LPS groups. At time zero, there were no significant dif-
ferences in the number of leukocytes adherent to the venular wall
among these groups, and in the control group there was only a
slight increase in cellular adherence to 1.72 ± 0.71 cells/200 μm
by the end of the observation period. In contrast, values in the
LPS group showed an impressive linear increase over time, from
5.00 ± 0.83 cells/200 μm at 20 min after LPS infusion was
initiated, to a high value of up to 12.72 ± 1.41 cells/200 μm at
60 min, representing an increase of more than fifteen-fold of the
time zero value. Treatment with Rb1, Rg1, or R1 simultaneously
with LPS resulted in attenuation of LPS-induced adhesion of
leukocytes to the venular wall, albeit with some variation in the
time course for each individual saponin, wherein the significant
inhibition of R1 started 20 min after LPS infusion, Rb1 started
40 min after LPS infusion, and Rg1 started only after 60 min. By
contrast, administration of Rb1, Rg1, or R1 alone had no effect
on leukocyte adhesion in comparison with control.
The time course of leukocyte emigration from the mesenteric
venular wall is summarized in Fig. 3. No emigrated leukocytes
Fig. 5. Effect of Rb1, Rg1, and R1 on the concentration of the cytokines TNF-α and IL-6 in plasma induced by LPS infusion for 60 min in vivo. The dosage of LPS was
2 mg/kg/h and the dosage of Rb1, Rg1, and R1 was 5 mg/kg/h. There were 6 rats in each group. Data are expressed as mean ± S.E. ⁎ P b 0.05 vs. control group,
#
P b 0.05 vs. LPS group. The concentrations of TNF-α and IL-6 increased markedly in response to LPS compared to the control group. Rb1, Rg1, and R1 treatment
inhibited the IL-6 increase, but had no effect on the release of TNF-α following LPS stimulation. Rb1, Rg1, or R1 treatment alone had no effect on the concentrations
of TNF-α and IL-6 in plasma.
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K. Sun et al. / Life Sciences 81 (2007) 509–518
515
Fig. 6. Effects of Rb1, Rg1, and R1 on the mean fluorescence intensity of the adhesion molecule CD11b on neutrophils exposed to LPS (2 μg/ml). The concentrations
of the saponins used in each condition are indicated in the figure. There were 6 rats in each group. Data are expressed as mean ± S.E. ⁎ P b 0.05 vs. control group,
#
P b 0.05 vs. LPS group. Expression of CD11b increased markedly after LPS stimulation compared to the control group, and Rb1 and R1 attenuated the increased
expression of CD11b at concentrations of 1.0 mg/ml and 0.2 mg/ml, respectively. Rg1 had no observable effect on CD11b expression. At the three concentrations
tested, Rb1, Rg1, or R1 alone had no effect on CD11b expression by neutrophils.
were noted at time zero in any instance, and only a few, if any,
emigrated leukocytes were observed over the entire observation
period for the control group. In contrast, the number of leukocytes emigrated from the venular wall increased profoundly
following LPS infusion, reaching 3.20 ± 0.73 cells /200 μm at
60 min, a four-fold increase compared to the control. This LPSinduced increase in the number of emigrated leukocytes was
significantly attenuated by treatment with R1, yielding a value
of 1.20 ± 0.37 cells/200 μm at 60 min. However, these effects
were not found with Rb1 or Rg1. Similarly, administration of
Rb1, Rg1, or R1 alone had no effect on leukocyte emigration
when compared with control.
Fig. 4 shows the percentage of degranulated mast cells observed along mesenteric venules 60 min after LPS infusion. The
percentage of degranulated mast cells in the control group was
19.8± 8.1%, representing spontaneous mast cell degranulation.
The number of degranulated mast cells increased to 49.4± 7.3% at
60 min after LPS infusion. Treatment with Rb1 and R1 significantly inhibited LPS-induced degranulation of mast cells (13.9 ±
3.8% and 23.8± 4.1%, respectively, at the end of the observation
period). Rg1 also appeared to exert some attenuation on LPSinduced degranulation (30.1 ± 7.4%, at the end of observation), but
this value was not statistically significant. However, Rb1, Rg1, or
R1 alone did not contribute to mast cell degranulation.
The concentrations of the cytokines TNF-α and IL-6 in
plasma are presented in Fig. 5. In the control group, the concentrations of TNF-α and IL-6 were 24.51 ± 5.57 and 22.72 ±
8.67 ng/l, respectively. The concentrations of the two cytokines
were enhanced dramatically by LPS stimulation to 493.97 ±
192.29 and 741.53 ± 126.86 ng/l, respectively. Treatment with
Rb1, Rg1, and R1 inhibited the LPS-induced production of IL-6
markedly. In contrast, none of them had effects on the release of
TNF-α. Meanwhile, Rb1, Rg1, and R1 alone had no effects on
the concentrations of TNF-α and IL-6 in plasma.
The percentages of both CD11b and CD18 positive cells
remained almost identical in all instances (data not shown).
However, if the expression of these adhesion molecules was
represented as fluorescence intensity (Figs. 6 and 7, respectively),
Fig. 7. Effects of Rb1, Rg1, and R1 on the mean fluorescence intensity of the adhesion molecule CD18 expressed by neutrophils exposed to LPS (2 μg/ml). The
concentrations of the saponins used in each condition are indicated in the figure. There were 6 rats in each group. Data are expressed as mean ± S.E. ⁎ P b 0.05 vs.
Control group, # P b 0.05 vs. LPS group. Expression of CD18 increased markedly after LPS stimulation compared to the control group, and Rb1 attenuated the
expression of CD18 at concentrations of 1.0 mg/ml and 2.0 mg/ml, while R1 attenuated the expression of CD18 at all three concentrations examined. Rg1 had no effect
on the expression of CD18. At the three concentrations tested, Rb1, Rg1, or R1 treatment alone had no effect on CD18 expression by neutrophils.
Author's personal copy
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K. Sun et al. / Life Sciences 81 (2007) 509–518
differences became distinct. The fluorescence intensity of CD11b
was enhanced dramatically by LPS stimulation, (247.9 ± 6.9,
1.8-fold increase over control), and the LPS enhanced expression
of CD11b by neutrophils was significantly inhibited by Rb1 and
R1 in a dose-dependent fashion, more prominently with R1. The
expression of CD18 by neutrophils exhibited a trend similar
to CD11b. After LPS stimulation, the fluorescence intensity
increased markedly, and the LPS-induced expression of CD18
was significantly reduced by Rb1 in a dose-dependent manner
(176.8 ± 10.3, 1.0 mg/ml and 138.9 ± 12.7, 2.0 mg/ml). Pretreatment with R1 resulted in a more striking effect on LPS-induced
expression of CD18 (119.6 ± 7.9, 0.2 mg/ml), although not in a
dose-dependent manner as for CD11b. It is noteworthy that Rg1
did not exhibit any significant effect on LPS-induced expression
of either CD11b or CD18 by neutrophils. On the other hand, at
the three concentrations examined, Rb1, Rg1, or R1 treatment
alone had no effect on the expression of CD11b and CD18 by
neutrophils.
The production of intracellular H2O2 by neutrophils is illustrated in Fig. 8, which is represented by the fluorescence
intensity of DCF. LPS stimulation markedly enhanced H2O2
production (191.0 ± 21.9) compared with the control (100.9 ±
19.5). Rg1 and R1 were able to significantly depress DCF
fluorescence intensity, while Rb1 had no significant effect
on H2O2 release. Although the production of intracellular ·O2−,
represented by the fluorescence intensity of EB was also apparently increased by LPS stimulation compared to the control
(194.6 ± 13.4 vs. 142.6 ± 13.4), none of the experimental saponins were found to have any statistically significant effect on
the LPS-induced elevation of intracellular ·O2− production (data
not shown). Still, Rb1, Rg1, or R1 treatment alone had no effect
on H2O2 and ·O2− release by neutrophils.
After LPS infusion for 1 h, AP in plasma increased significantly compared with the control group (168.33 ± 4.33 vs.
119.00 ± 14.73 unit/l, respectively). However, none of the experimental saponins showed a statistically significant effect on
the increase of AP expression induced by LPS (data not shown),
and administration of Rb1, Rg1, or R1 alone had no effect on
the release of AP.
Discussion
The goal of the present study was to evaluate the effects
of Rb1, Rg1, and R1 on LPS-induced microcirculatory disturbance, including the effects of these three saponins on the
expression of the adhesion molecules CD11b/CD18, and the
release of leukocyte H2O2 and ·O2−. The results of in vivo rat
experiments demonstrated that both Rb1 and R1 attenuated LPSinduced leukocyte adhesion and mast cell degranulation; R1 also
reduced leukocyte emigration, while Rg1 was effective only in the
inhibition of leukocyte adhesion. In addition, all three saponins
depressed secretion of cytokines in plasma. In vitro expression of
CD11b/CD18 showed the same tendencies as observed in the
in vivo experiment. Consequently, R1 and Rb1, but not Rg1, are
likely useful for any application intending to attenuate LPS-elicited
leukocyte recruitment and mast cell degranulation. On the other
hand, Rg1 as well as R1 were observed to blunt the release of
neutrophil H2O2 induced by LPS, implying that R1 possesses a
more broad spectrum potential and that Rg1 exhibits a more specific effect, as far as the parameters examined in the present study.
The recognition that microcirculatory disturbance is implicated in various cardiovascular diseases has resulted in an intense
effort to identify the cellular and molecular events in this process.
Rats intravenously infused with low concentrations of LPS have
been a widely used animal model for investigating these events
(Hoffmann et al., 2000; Woodman et al., 2000). Results using this
model cover a range of phenomena that occur in the LPS-induced
microvascular response, including recruitment of leukocytes in
the venules, enhanced expression of adhesion molecules by both
endothelium and neutrophils, elevated production of free radicals,
and an increase in the number of degranulated mast cells. Based
on these observations, some approaches have been proposed to
impede LPS-induced injury, such as the use of antibodies to
adhesion molecules (Moreland et al., 2002) or free radical
Fig. 8. Effects of Rb1, Rg1, and R1 on the release of neutrophil H2O2 induced by LPS (2 μg/ml). The concentration of each of the saponins was 1.0 mg/ml. There were
6 rats in each group. Data are expressed as mean ± S.E. ⁎ P b 0.05 vs. control group, # P b 0.05 vs. LPS group. H2O2 production increased markedly after LPS
stimulation compared to the control group. Rg1 and R1 significantly reduced H2O2 release. Rb1 did not affect the increased production of H2O2 induced by LPS. Rb1,
Rg1, or R1 treatment alone had no effect on H2O2 release by neutrophils.
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K. Sun et al. / Life Sciences 81 (2007) 509–518
scavengers in the postcapillary venules (Tsuji et al., 2005). However, much more work is required before any of these approaches
can be employed in clinical practice due to their complexity. On
the other hand, a number of traditional Chinese herbal medicines
have long been effectively employed in China to improve blood
circulation and ameliorate pathological hemostasis. The herbal
formulation PN constitutes the major component of some of these
Chinese medicines, such as Myakuryu (a preparation that contains
the herbal preparation PN as a major component) and Cadiotonic
pills which are used clinically in China and other Asian nations.
Over the past several years, a number of publications have dealt
with the mechanisms that underlie the clinical effectiveness of the
herbal preparation PN or PNS. However, many issues remain
unresolved. In particular, PNS consists of more than 30 different
types of saponins, and the role played by each of these saponins
in the attenuation of microcirculatory disorder is as yet poorly
understood.
We have previously reported that PNS could inhibit the
LPS-induced adhesion of leukocytes to the venular wall in rat
mesentery (Sun et al., 2006). The present in vivo experiments
demonstrate that the three major saponins of PNS, Rb1, Rg1, and
R1, are specifically capable of attenuating the LPS-induced
adhesion of leukocytes. Furthermore, this study revealed that
R1, Rb1, and Rg1 exhibit attenuating effects at different time
points following LPS infusion and show effects to different
degrees, implying that the potentials of these three saponins are
distinct. It is likely that the differences in the chemical structures
of the saponins shown in Fig. 1 are responsible for their distinct
potential. If valid, these results may be useful in designing a drug
to attenuate leukocyte recruitment induced by LPS.
We have previously reported that PNS can inhibit the LPSinduced expression of CD11b/CD18 by neutrophils in rats (Sun
et al., 2006). The present study revealed a profound inhibitory
effect on LPS-induced neutrophil expression of CD11b/CD18 by
R1 and Rb1, while Rg1 showed no effect. R1 exhibited a more
intense inhibitory effect on the expression of either CD11b or CD18
than Rb1. The reason for the failure of Rg1 to display any inhibitory
effect in the present work is not clear. One possible explanation is
that the inhibitory effect of Rg1 on leukocyte adhesion to the
venular wall detected in the in vivo experiment is mediated by
adhesion molecule(s) other than CD11b/CD18. As such, the effects
of Rb1, Rg1, and R1 on the expression of other adhesion molecules
warrant investigation. The mechanism whereby R1 and Rb1 exert
an inhibitory effect on the enhanced expression of CD11b/CD18
induced by LPS is not clear, and further studies are required.
Accumulating evidence demonstrates that ROS induce
CD11b/CD18 upregulation and CD11b/CD18 mediated neutrophil adhesion (Fraticelli et al., 1996). Since none of the saponins
tested in this study exhibited any effect on the release of ·O2−, and
since only Rg1 and R1 were found to reduce the concentration of
H2O2, the engagement of free radicals in the protective effect of
these saponins on the expression of CD11b/CD18 might be
excluded from consideration as a possible mechanism. Nevertheless, the effect of Rg1 and R1 on the release of neutrophil H2O2
supports the idea that these two saponins, as well as PNS, counteract the microcirculatory dysfunction induced by LPS via diverse pathways, taking into account the fact that reactive oxygen
517
species derived from adherent neutrophils is a potential mediator
for initiation of endothelial injury (Pober and Min, 2006).
It has been reported that mast cells are activated and degranulated by LPS (Iuvone et al., 1999), and this pathway is also
activated in the presence of ROS (Wan et al., 2001). When
degranulated, mast cells release histamine and TNF-α, and initiate
the cytokine cascade, which leads to the expression of vascular
cell adhesion molecule-1 (VCAM-1) and E-selectin on endothelial cells and subsequent neutrophil-induced injury (Frangogiannis et al., 1998; van Haaster et al., 1997). The inhibitory effect of
PNS on mast cell degranulation was previously identified from
the results of experiments with the compound Myakuryu (Han
et al., 2006). The results in the present study further confirm this
effect, and concomitantly demonstrate that Rb1, Rg1, and R1
depress the elevated concentration of IL-6 in plasma induced by
LPS, indicating that the experimental saponins could decrease the
action of cytokines on the blood vessel wall.
Many studies have shown that LPS strongly activates intestinal alkaline phosphatase (AP) expression, indicating that AP
is capable of detoxifying LPS through dephosphorylation
(Koyama et al., 2002). Administration of AP attenuated the
excessive LPS-induced inflammatory response and prevented a
lethal outcome in mice (Verweij et al., 2004). Our results show
that AP in the plasma increased markedly after 1 h of LPS
infusion, and administration of Rb1, Rg1, or R1 had no effect on
the elevated AP, indicating that detoxification of LPS may not be
implicated in the mechanism by which the saponins ameliorate
LPS-induced microcirculatory disturbances.
In conclusion, the results of the present study demonstrate that
Rb1, R1, and Rg1 are capable of attenuating the adhesion of
leukocytes induced by LPS to differing extents, and this effect may
contribute by an as yet unidentified mechanism to the capacity of
these agents to depress the expression of CD11b/CD18. The
identification of an inhibitory effect on mast cell degranulation by
Rb1 and R1, and on the release of H2O2 by Rg1 and R1 reflect the
diversity of roles that PNS may play in the amelioration of microcirculatory disorders which occur in response to LPS stimulation.
Acknowledgement
The authors would like to thank Michael Allen McNutt, Ph.D.
for valuable discussions. This study was supported financially
by the Tianjin Tasly group, Tianjin, China. The grant number was
T 03302005.
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