mediator inducing VCAM-1 expression [24, 25], the role of ROS production in TNF-α-induced VCAM-1 expression was investigated. As shown in Figure 5H-5J, 2 h
pretreatment with the antioxidant NAC or the NADPH oxidase inhibitors DPI or APO
significantly attenuated TNF-α-induced VCAM-1 expression in a
concentration-dependent manner. In addition, as shown in Figure 5K, 2 h pretreatment with antioxidants (NAC, DPI, or APO) partly inhibited TNF-α-induced JNK
phosphorylation, the effects being similar to that of viscolin. These results suggest
that NADPH oxidase-derived ROS production plays a critical role in TNF-α-induced
VCAM-1 expression.
Viscolin reduces the adhesion of monocytes to TNF-α-treated HUVECs
To explore the effects of viscolin on the endothelial cell-leukocyte interaction, we
examined the adhesion of U937 cells to TNF-α-activated HUVECs. As shown in Fig. 6,
control confluent HUVECs (panel C) incubated with U937 cells for 1 h showed minimal
binding, but adhesion was substantially increased when the HUVECs were pretreated
with TNF-α for 6 h (panel T). Pretreatment of HUVECs with viscolin for 24 h (panel
T+Vis) reduced the number of U937 cells adherent to TNF-α-treated HUVECs by
46±4% compared to TNF-α alone. The involvement of VCAM-1 in the adhesion of U937
cells to TNF-α-treated HUVECs was examined by pretreatment of the cells with
anti-VCAM-1 antibody. When HUVECs were pretreated with 1 µg/mL (panel VCAM-1
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Ab-1) or 2 µg/mL (panel VCAM-1 Ab-2) of anti-VCAM-1 antibody for 1 h, then
incubated with TNF-α, the binding of U937 cells to HUVECs was significantly lower
than that to non-antibody-treated TNF-α-stimulated cells, showing that VCAM-1 plays a
major role in the adhesion of U937 cells to TNF-α-treated HUVECs. The adherence of TNF-α-treated U937 cells to HUVECs was also inhibited by 1 h pretreatment with 10
µM PD98059 (panel T+PD), SP600125 (panel T+SP), SB203580 (panel T+SB), or
parthenolide (panel T+Par). Similarly, the adherence of U937 cells to TNF-α-treated
HUVECs was also inhibited by 2 h pretreatment with antioxidants (NAC, DPI, or APO).
Viscolin reduces VCAM-1 protein expression in the thoracic aorta in TNF-α-injected
mice
To determine the effect of viscolin on VCAM-1 expression in vivo, mice were
injected with viscolin for 5 days prior to injection with TNF-α for 3 days, then
immunohistochemical staining was performed to detect the expression of VCAM-1 on
serial section of thoracic aorta, using vWF as an endothelial cell marker. As shown in
Figure 7, in the control (panel C) and viscolin-treated (Vis) groups, no VCAM-1
staining was seen on the vascular wall, while, in the TNF-α-treated group (panel TNF-α), strong VCAM-1 staining was seen on the luminal surface. In contrast,
pre-administration of viscolin resulted in weak VCAM-1 staining in the TNF-α-treated animals (panel TNF-α+Vis).
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Discussion
In the present study, we demonstrated that viscolin treatment reduced VCAM-1
expression both in vitro in TNF-α-stimulated HUVECs and in vivo in the thoracic aorta of TNF-α-treated mice. Viscolin also inhibited the binding of the human monocytic cell
line U937 to TNF-α-stimulated HUVECs. These effects were inhibited by SP600125, a
JNK inhibitor, or parthenolide, a NF-κB inhibitor, showing that they were partly
mediated through inhibition of JNK phosphorylation and NF-κB activation. In addition,
viscolin attenuated the increase in VCAM-1 mRNA expression and VCAM-1 promoter activity induced by TNF-α. Furthermore, viscolin had a scavenging effect on the
generation of ROS as well as on the decreased NADPH oxidase activity.
Viscolin, isolated from Viscum coloratum, was chosen for testing, as Viscum
coloratum has long been used in traditional Chinese medicine to treat inflammatory
diseases. Antioxidative and anti-inflammatory actions are two of the pharmacological
properties proposed to underlie its beneficial effects [7-9]. A partially purified fraction
from the chloroform extract of V. coloratum (PPE-SVC) has been shown to inhibit the
generation of superoxide anions by formyl-L-methionyl-L-leucyl-L-phenylalanine
(fMLP)-activated human neutrophils, and purified viscolin, a major active component of
PPE-SVC, inhibits the generation of superoxide anion and the release of elastase in
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fMLP-activated human neutrophils [7]. Viscolin suppresses ROS and nitric oxide (NO)
generation in leukocytes and microglial cells, and, in addition, attenuates
pro-inflammatory cytokine production [9]. The present study is the first to report that
viscolin strongly reduces levels of VCAM-1 mRNA and protein in TNF-α-treated HUVECs. The present results also showed that viscolin reduced TNF-α-induced
VCAM-1 promoter activity.
Our results demonstrated that TNF-α induced time-dependent phosphorylation of
MAPKs (ERK1/2, JNK, and p38) and that the increases in VCAM-1 expression and
U937 cell adhesion induced by TNF-α were inhibited by PD98059, SP600125, or
SB203580. These results show that activation of MAPKs is necessary for
TNF-α-induced VCAM-1 expression in HUVECs. Consistent with these findings,
TNF-α-induced VCAM-1 expression in human tracheal smooth muscle cells requires
activation of MAPKs [26]. Furthermore, our results demonstrated that viscolin
inhibited the TNF-α-induced phosphorylation of JNK, but not that of ERK1/2 or p38
(Figure 3), suggesting that the inhibitory effect of viscolin on VCAM-1 expression is
mediated, in part, by JNK inhibition. Since a previous study showed that ROS
regulate both protein kinases and protein phosphatases [27], one of our future aims is
to determine the protein phosphatases involved in the dephosphorylation of JNK that
are regulated by viscolin. In addition, our results also showed that viscolin inhibited
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the TNF-α-induced increase in VCAM-1 mRNA levels. Although we cannot rule out
the possibility that viscolin may affect the stability of VCAM-1 mRNA, viscolin was
found to inhibit the TNF-α-induced promoter activity of VCAM-1 (Figure 2D). These
results suggest that viscolin attenuates VCAM-1 expression induced by TNF-α, at
least in part, through a transcriptional mechanism.
Several lines of evidence indicate that TNF-α induces ROS production in
endothelial cells [10, 24, 25, 28]. Consistent with these previous results, our study
showed that it rapidly induced ROS production and that this was inhibited by the NADPH oxidase inhibitors DPI and APO. These results suggest that TNF-α induces
ROS production via activation of NADPH oxidase. ROS appears to be a second
messenger in the TNF-α-induced signal transduction pathway that regulates VCAM-1
expression [10, 24, 25, 28]. In our study, antioxidants (NAC, DPI, or APO) inhibited the TNF-α-induced increase in VCAM-1 expression (Figure 5H-J) and U937 cell
adhesion (Figure 6A), showing that ROS mediated the effects of TNF-α on VCAM-1
expression. In addition, preincubation with viscolin effectively attenuated the ROS
production induced by TNF-α in HUVECs. Moreover, pretreatment with antioxidants (NAC, DPI, APO) inhibited TNF-α−induced JNK phosphorylation to a similar extent
as viscolin. These results suggest that viscolin inhibits TNF-α−induced VCAM-1
expression via its antioxidative properties. Furthermore, we demonstrate that viscolin
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inhibited NADPH oxidase activity and p47expression in the membrane fraction of TNF-α−treated HUVECs. Because of their chemical structure, a benzene ring with
adjacent methoxy-hydroxyl groups, flavonoids are potent inhibitors of NADPH
oxidase activity [29]. As the chemical structure of viscolin (Figure 1) is similar to that
of flavonoids, it may have a similar inhibitory effect on NADPH oxidase activity.
Future studies are necessary to clarify the role of viscolin on NOX activity as DPI and
apocyanin have been reported to inhibit NOX activity as well as affect other reactive
species and enzymes.
The VCAM-1 gene promoter contains consensus binding sites for AP-1 and NF-κB [21, 22]. Our results showed that the binding activity of NF-κB and AP-1 was
activated by TNF-α and that pretreatment with viscolin significantly inhibited the
TNF-α-induced increase in binding activity of NF-κB, but not that of AP-1. In
addition, several reports have shown that natural products with antioxidant activity inhibit the TNF-α−induced activation of redox-sensitive NF-κB [10, 24, 25, 28].
Pretreatment with an NF-κB inhibitor suppressed the TNF-α-induced increase in
VCAM-1 expression and U937 cell adhesion, suggesting that viscolin attenuates VCAM-1 expression via a reduction in NF-κB binding activity. Our results showed
that viscolin and the antioxidants NAC, DPI, and APO significantly attenuated NF-κB
binding activity and NF-κB p65 translocation, and that these effects may be due to its
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antioxidative activity. Viscolin has anti-inflammatory and antioxidative properties
based on the above findings. Because atherosclerosis is a chronic inflammatory
disease [1,2], viscolin may be beneficial for the prevention of inflammation and
atherosclerosis.
In conclusion, our study demonstrated that viscolin reduces VCAM-1 expression
under inflammatory conditions both in vitro and in vivo. Our results show that the
inhibitory effect on VCAM-1 expression is partly mediated by inhibition of JNK
phosphorylation, NF-κB activation, and ROS production. Our results demonstrated
the anti-inflammatory and antioxidative effects of viscolin, an active component of V.
coloratum, on endothelial cells and suggested that this compound may provide a
chemical backbone for the development of therapeutic agents.
Acknowledgements
This work was supported by research grants from the National Science Council
(NSC-99-2320-B-002-0220MY3) and the Cooperative Research Program of the NTU
and CMUCM (99F0080-303), Taiwan, ROC.