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Introduction
Heart failure remains one of the leading causes of death
in all industrialized nations, and the quest for novel
treatment options has recently directed attention to cardiac
myocyte injury as a promising target, since this approach might
provide the opportunity to prevent disease progression
rather than treating established cardiomyopathy and heart
failure[1,2]. It has been recently shown that beta-adrenergic
agonists can induce apoptosis in cultured neonatal cardiac
myocytes[3,4], suggesting that it might be one of the factors
involved in myocardial cell injury in heart
failure in vivo.
Silibinin (Figure 1), a polyphenolic flavanoid, exhibits
potent antioxidant activity[5], in addition to
hepatoprotec-tive[6,7] and anti-inflammatory
effects[8]. Besides these, silibinin was found to have cytoprotective
effects[9]. Numerous studies show that ageing is associated with increased rates of
stress-induced apoptosis[10], and the cumulative effects of
cell loss have been implicated in various diseases including
neurodegeneration, retinal degeneration, cardiovascular
disease and frailty[11_13]. Therefore, searching for the active
compounds from Chinese herbal medicines which inhibit
disease-associated apoptosis or protect cells from various
causes of death is the aim of our study.
Previous studies pertaining to the components of the
signaling system in cardioprotection have implicated a role
for tyrosine kinases[14]. It has been increasingly apparent
that G proteins are involved in the regulation of cell growth
and differentiation as well as receptor protein tyrosine
kinases (RPTK)[15]. At the intracellular face of the cell
membrane, the exchange of GTP to GDP increases the
affinity of the small G protein Ras for c-Raf, which then
translocates to the membrane[16,17]. In RPTK-linked signaling,
adaptor proteins such as Shc and growth factor receptor bound 2
(Grb2) interact directly with the phophorylated receptors to
promote activation of Ras and Raf. Grb2 binds to the
phosphotyosine through its SH2 domains and constitutively
associates with polyproline domains in the GEF Sos.
Binding of Grb2 to an RPTK thus places Sos in the vicinity of Ras
GDP[18_20].
A mediator of G-protein-signaling that has become the
focus of recent investigations is the protein kinase C (PKC)
family which is comprised of at least 12 members that share
structural homology and induce a great variety of
intracellular responses when activated by lipid products of
phospholipase C or D activity[21,22].
Biochemical and genetic studies in various cell systems
have demonstrated that Raf-1 functions downstream of
activated tyrosine kinases and Ras, and upstream of MAPK
kinase (MEK) and mitogen-activated protein kinase
(MAPK)[23,24]. Growing evidence suggests that modulation
of complex network of MAPK signaling cascades could be a
rewarding approach to treat cardiomyocyte hypertrophy and
heart failure[25]. The MAPK are elements of 3-tiered protein
kinase cascades and basically comprise 3 subfamilies: the
extracellularly responsive kinases (ERK), the c-Jun
N-terminal kinases (JNK), and the p38 MAPK. While the ERK are
particularly implicated in growth-associated responses, the
latter 2 groups are generally activated by cytotoxic stress
factors[26].
In the present study, we demonstrate whether the
tyrosine kinase pathway participates in silibinin-protected rat
cardiac myocyte apoptosis, and whether the activity of
upstream PKC is required for the increase of Ras and Raf-1.
Among the downstream targets Ras and Raf-1 are of MAPK
members. We tried to examine the expression of MAPK in a
beta-adrenergic agonist, isoproterenol, in stimulation-
induced injury of cultured rat neonatal cardiac myocytes
after silibinin treatment.
Materials and methods
Chemical reagents Silibinin (Lot 0856_9902) was
obtained from the Beijing Institute of Biologic Products (Beijing,
China). The purity of silibinin was measured by HPLC and
was determined to be about 99%. Silibinin was dissolved in
DMSO to make a stock solution. The DMSO concentration
was kept below 0.1% in all the cell cultures and did not exert
any detectable effects on cell growth or cell death.
Genistein (a PTK inhibitor), manumycin A (a Ras
inhibitor), GW5074 (a Raf-1 inhibitor) and staurosporine (a
PKC inhibitor) were purchased from Enzyme Systems (Livermore, CA, USA). PD98059 (an ERK inhibitor), SP600125
(a JNK inhibitor) and SB203580 (a p38 MAPK inhibitor) were
purchased from Calbiochem (La Jolla, CA, USA).
Fetal bovine serum (FBS) was from Tianjin TBD (Tianjin,
China). Dulbecco's modified Eagle's medium (DMEM) was
from Hyclone (Logan, UT, USA). Murine polyclonal
antibodies against p-ERK, p-JNK and p-p38, rabbit polyclonal
antibodies against Ras, Raf-1, Grb2, ERK, JNK, p38 and
horseradish peroxidase (HRP)-conjugated secondary antibody
(goat anti-rabbit and goat anti-mouse) were obtained from
Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Cell culture Primary ventricular cardiac myocytes were
prepared as previously described[27]. Briefly, the hearts from
1_2 d old Sprague-Dawley rats were removed, the ventricles
were pooled, and the ventricular cells were dispersed by
digestion with collagenase II. The cells were pre-plated for 1
h to enrich the culture with myocytes (90%_95% of cells
after this step). Then the cells were cultured in media
consisting in high glucose DMEM, 2 mmol/L L-glutamine
(GIBCO, Grand Island, NY, USA), penicillin (100 U/mL) and
streptomycin (100 μg/mL), 10% FBS at 37
oC and 5% CO2 in a humidified atmosphere. The cells were added with silibinin
and isoproterenol after 48 h with 5% FBS.
Cell growth assay The cytotoxic effects of isoproterenol
on cardiac myocytes were measured using MTT assay as
described[28]. The cells were dispensed in 96-well
flat-bottomed microtiter plates (Nunc, Roskilde, Denmark) at a
density of 5×105 cells/well. After 48 h incubation, they were
treated with isoproterenol (10 μmol/L) and/or silibinin at
various concentrations for 48 h. The viability was calculated as
follows:
Viability
(%)=(A490,sample_A490,
blank)/(A490,
control_A490, blank)×100
Terminal deoxynucleotidyl transferase-mediated dUTP
nick end-labeling (TUNEL) assay The TUNEL assay was
used for the detection of DNA strand breaks. The detection
was carried out according to the instructions of TACS2
TdT-DAB in situ apoptosis detection kit. Briefly, the cells were
rinsed once with PBS and fixed in 3.7% buffered
formaldehyde at room temperature for 10 min. The fixed sections were
pretreated with 10% H2O2, and end-labeling was performed
with TdT labeling reaction mix at 37 oC for 1 h. Nuclei
exhibiting DNA fragmentation were visualized by incubation
in 3,3'-diamino benzidine (DAB) for 7 min. Finally, the sections
were counterstained with methyl green, and observed by
light microscopy. The nuclei of the apoptotic cells were
stained dark brown; TUNEL-positive cardiac myocytes were
determined by randomly counting 100 cells.
PKC activity assay PKC activity assay was carried out
according to the instructions of the PepTag non-radioactive
protein kinase C assay kit (Promega, Madison, WI, USA).
Briefly, the cells were washed once with PBS and lysed in
lysis buffer, including 20 mmol/L Tris-HCl, 0.5 mmol/L EGTA,
2 mmol/L EDTA, 2 mmol/L phenylmethanesulfonyl fluoride
(PMSF), and 10 mg/L leupeptin (pH 7.5). Assays were then
performed at 30 oC in a total volume of 25
µL containing 5 µL PKC reaction 5× buffer, 5 µL PLSRTLSVAAK peptide, 5 µL
PKC activator, 1 µL peptide protection solution, and 9 µL
sample. Reactions were initiated by the addition of the
9 µL sample and terminated after 30 min by incubation of the
reaction mixture at 95 oC for 10 min. After adding
1 µL of 80% glycerol, each sample was separated by 0.8 % agarose gel
electrophoresis at 100 V for 15 min.
Western blot analysis After incubation for 48 h, both
adherent and floating cardiac myocytes were collected.
Western blot analysis was carried out as previously
described[29] with some modifications. The cells were lysed on ice in lysis
buffer [50 mmol/L HEPES (pH 7.4), 1% Triton X-100, 2
mmol/L sodium orthovanadate, 100 mmol/L sodium fluoride,
1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L PMSF],
supplemented with proteinase inhibitors (100 µg/mL aprotinin, 10
µg/mL leupeptin, and 100 µg/mL pepstatin) for 1 h. The
protein concentration was determined by Folin assay. The
lysate was centrifugated at 16 000×g at 4
oC for 10 min; equal amounts of total proteins were mixed in 2× loading buffers
[50 mmol/L Tris-HCl (pH 6.8), 2% SDS, 10%
2-mercapto-ethanol, 10% glycerol, and 0.002% bromphenol blue], boiled
for 5 min, and run on a 12% or 15% SDS-PAGE. Proteins were
electrotransferred onto nitrocellulose membranes and
detected with antibodies against Ras, Raf-1, Grb2, MAPK and
b-actin, followed by the addition of HRP-conjugated
secondary antibody and 3,3'-DAB as the HRP substrate.
Statistical analysis All data represented at least 3
independent experiments and were expressed as mean±SD,
unless otherwise indicated. One-way ANOVA (SPSS 11.0
software, SPSS, Chicago, IL, USA) was used for statistical
analysis, and P<0.05 was considered statistically significant.
Results
Involvement of tyrosine kinase pathway in the protective
effect of silibinin Viability and TUNEL assays were carried
out to confirm the involvement of the tyrosine kinase
pathway in the silibinin-treated cardiac myocytes with
isoproterenol exposure. Treatment with 40 µmol/L PTK inhibitor
(genistein), 5 µmol/L Ras inhibitor (manumycin A) or 10
nmol/L Raf-1 inhibitor (GW5074) significantly reversed the
protective effects of silibinin (Figure 2; Table 1).
Ras, Raf-1 and the adaptor protein, Grb2, decreased after
incubation with 10 µmol/L isoproterenol for 48 h, whereas
silibinin reversed their expression (Figure 4), but after
incubation with silibinin alone, the expression of Ras, Raf-1 and
Grb2 had no changes, indicating that silibinin could activate
the tyrosine kinase pathway when the pathway was inhibited.
PKC activity increased dose-dependently during
silibinin-treatment Very recently, clear evidence has been
presented that PKC acts to increase Ras GTP loading, and
that the activation of ERK by PKC is Ras
dependent[30], thus PKC activity was assayed. The specific fluorescent substrate
phosphorylation was observed by treatment with
isoproterenol and silibinin. Silibinin dose-dependently increased PKC
activity compared to isoproterenol, and PKC activity had no
changes after treatment with silibinin alone (Figure 3).
Treatment with 100 nmol/L staurosporine significantly
decreased the expression of Ras, Raf-1 and Grb2 (Figure 4).
It was suggested that silibinin prevented
isoproterenol-induced rat cardiac myocyte apoptosis through the tyrosine
kinase pathway after upregulation of PKC activity.
Effects of inhibitors of ERK, p38 and JNK on protective
effects of silibinin MAPK cascade is an important signaling
modulation pathway that propagates extracellular
stimulation into intracellular responses and regulates cell growth,
differentiation, and apoptosis[31]. To determine whether
MAPK family was involved in silibinin-treatment, 10
μmol/L ERK inhibitor PD98059 (Figure 5A), 10 μmol/L p38 MAPK
inhibitor SB203580 (Figure 5B) and 10 μmol/L JNK inhibitor
SP600125 (Figure 5C) were administered. Cardiac myocytes
were pretreated with 10 μmol/L PD98059, SB203580 and
SP600125 for 60 min, and then cultured with silibinin and
isoproterenol for 48 h. The protective effect of silibinin was
significantly reduced by PD98059, while isoproterenol-
induced apoptosis was reversed by SB203580 and both of
them were unaffected by SP600125. TUNEL-positive myocardiac cells were increased after inhibitors added, which
suggests that the inhibitors could induce myocardiac cells
apoptosis (Table 2).
ERK phosphorylation was increased by silibinin
treatment and was dependent on the tyrosine pathway and PKC
activity After cardiac myocytes were exposed to silibinin or
isoproterenol for 48 h, expression of phosphor-ERK increased
(Figure 6A), whereas the expression of phosphorylated p38
decreased compared to isoproterenol alone treatment (Figure
6B), and JNK phosphorylation (Figure 6C) did not change.
The expression of ERK and JNK as well as p38 MAPK
protein in a whole cell lysate did not change. Ras, Raf-1 and
PKC inhibitors caused decrease in ERK phosphorylation,
but they had no effect on p38 and JNK phosphorylation
(Figure 7). These results suggest that tyrosine kinase
pathway and PKC activity affected ERK phosphorylation after
treatment with silibinin in rat cardiac myocyte culture. Silibinin
had no effect on the activity of MAPKs alone (Figure 6).
Discussion
In response to stress stimulation, cells activate
biochemical pathways that allow adaptation to this stressful
environ-ment. However, with prolonged isoproterenol treatment, these
protective mechanisms may not be sufficient to maintain
normal cellular function, and cell injury and death
follow. Our previous study showed that silibinin could protect rat
cardiac myocytes from apoptosis. In this study, we have
demonstrated that genistein, manumycin A and GW5074
significantly reversed the protective effect of silibinin. In addition,
the PKC inhibitor, stauroporine, markedly reduced Ras and
Raf-1 expression after silibinin treatment. Within the MAPK
family members, only ERK was phosphorylated in silibinin
treatment and was dependent on the activity of Ras, Raf-1,
and PKC.
A central feature of signal transduction downstream of
both receptor and oncogenic tyrosine kinases is the
Ras-dependent activation of a protein kinase cascade consisting
of Raf-1, MEK, and ERK (MAP
kinases)[32]. It has been well reported that the family of small G proteins functions as an
important link between cell membrane receptors and
numerous signaling pathways[33]. Thus, the involvement of small G
proteins in cardiac death has become an area of growing
interest. Five subfamilies of small G proteins have been
described (Rho, Ras, ARF, Rab, Ran), of these, the role of
Ras and Rho in cardiac injury have been examined in most
detail[34]. Studies of various cell systems have shown that
Shc, Grb2, and Sos link activated tyrosine kinases. Potential
activators of Raf-1 include protein kinase and activated
Ras[35,36]. Our results show that after administration of
genistein, manumycin A and GW5074, the protective effects
of silibinin were suppressed markedly, indicating that the
tyrosine kinase signal cascade including PTK, Ras, and
Raf-1 was involved in the enhancement.
The PKC signaling system in cardioprotection has 2
components: (1) the molecules that are involved in PKC
signaling; and (2) the manner in which these molecules
interact with PKC[37,38]. Evidence from recent studies in
numerous cellular theaters suggests that PKC-mediated
cardiopro-tection is associated with dynamic modulation of signaling
complexes[39]. We further confirmed the PKC activity, which
was attenuated by isoproterenol, whereas the impaired
activity was reinforced by silibinin. Furthermore, the
expression of Ras, Raf-1 and Grb2 were reversed by the PKC
inhibitor stauroporine, indicating that PKC was activated in this
process, and the activities of Ras and Raf-1 were dependent
on PKC.
The Ras and Raf-1 proto-oncogene products are key
proteins in the transmission of a variety of intracellular
proliferation and differentiation signals. Raf-1 and Ras serve as
intermediates in these signaling pathways, connecting
upstream tyrosine kinases with downstream
serine/threonine kinases such as ERK and
MEK[23,24]. This phosphorylation cascade leads to the activation of transcription factors
involved in cell growth and differentiation. Raf-1-dependent
activation of ERK1 and ERK2 has been demonstrated in
wounded intestinal epithelial
monolayers[40], mechanically stretched rat cardiac
myocytes[41], and shear stress-exposed human endothelial
cells[42]. PKC can activate MAPK, which
in turn activates the gene transcription involved in cell
proliferation and differentiation. Growth factor-mediated
activation of the Raf-1_MAPK/ERK cascade may therefore involve
either Ras, PKC or both. Our results show that ERK inhibitor,
PD98059, in response to silibinin, inhibited the protective
effect, whereas the p38 MAPK inhibitor, SB203580,
significantly reversed isoproterenol-induced apoptosis; JNK
inhibitor, SP600125, had no effect. Western blot analysis
further confirmed that phosphorylation of ERK increased with
silibinin treatment, but phosphorylation of p38 decreased,
and that of JNK did not change, indicating that the
protective effect of silibinin required the activation of ERK. Adding
Ras, Raf-1 and PKC inhibitors caused a decrease in ERK
phosphorylation only, indicating that ERK phosphorylation
was dependent on the activity of the tyrosine pathway and
PKC.
Silibinin had no effect on the activity of the tyrosine
kinase pathway, PKC and MAPK alone, but could activate
them when they were inhibited, indicating that silibinin could
preserve the balance of kinases in cell homeostasis. Further
investigation is necessary to clarify the target of silibinin.
Taken together, the present study shows that silibinin
protects isoproterenol-induced apoptosis in rat cardiac
myocytes through the activation of PKC involving Ras,
Raf-1 and the phosphorylation of ERK.
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