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Acta Biochim Biophys |
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doi:10.1111/j.1745-7270.2008.00463.x |
Salidroside inhibits H2O2-induced
apoptosis in PC12 cells by preventing cytochrome c release and
inactivating of caspase cascade
Lei Cai1, Hua Wang2, Qin Li2, Yunfei Qian1, and Wenbing Yao1*
1 School of Life Science and Technology,
China Pharmaceutical University, Nanjing 210009, China
2 Yangtze River Pharmacy Group, Guangzhou Hairui
Pharmaceutical Company, Guangzhou 510663, China
Received: March 11,
2008
Accepted: June 17,
2008
This work was
supported by grants from the Teaching and Research Award Program for
Outstanding Young Teachers (No. 2002-383), the Program for New Century
Excellent Talents in University (No. NCET-04-0506) and the Traditional Chinese
Medicine Research Foundation of Science and Technology (No. 04-05ZP33)
*Corresponding
author: Tel, 86-25-83271218; Fax, 86-25-83271218; E-mail, [email protected]
We used a rat pheochromocytoma (PC12) cell
line to study the effects of salidroside on hydrogen peroxide (H2O2)-induced apoptosis. In PC12 cells, H2O2-induced apoptosis was accompanied by the down-regulation
of Bcl-2, the up-regulation of Bax, the release of mitochondrial cytochrome c
to cytosol, and the activation of caspase-3, -8 and -9. However, salidroside
suppressed the down-regulation of Bcl-2, the up-regulation of Bax and the
release of mitochondrial cytochrome c to cytosol. Moreover, salidroside
attenuated caspase-3, -8 and -9 activation, and eventually protected cells
against H2O2-induced apoptosis. Taken together, these
results suggest that treatment of PC12 cells with salidroside can block H2O2-induced apoptosis by regulating Bcl-2 family members
and by suppressing cytochrome c release and caspase cascade activation.
Keywords salidroside; hydrogen peroxide; apoptosis; PC12 cells
Alzheimer’s disease (AD) is a multifaceted neurodegenerative
disorder characterized by the progressive deterioration of cognition and
memory in association with widespread neuronal loss and the deposit of senile
plaques. To date, the cause and the mechanism by which neurons die as a result
of AD still remain unclear, yet several lines of evidence support the
involvement of apoptosis. Studies on post-mortem tissues have provided direct
morphological and biochemical evidence that some neurons in the brains of AD
patients degenerate via an apoptotic mechanism relating to the presence of DNA
damage, nuclear apoptotic bodies, and other markers of apoptosis [1,2]. These results suggest therapeutic strategies aimed at preventing
and delaying apoptosis might be a reasonable choice for the treatment of the
disease.
Hydrogen peroxide (H2O2), a
major source of reactive oxygen species, destroys neurons by inducing
apoptosis, which has implications for several biological and pathological processes,
including AD. H2O2 has been used in many
studies to trigger cell apoptosis [3,4]. Therefore, we used H2O2
to induce apoptosis in PC12 cells in present study.
Considerable efforts have been made to find natural substances with
neuroprotective potential, and attention has been focused particularly on
Chinese medicinal plants with nootropic effects. Some plants have been
used for thousands of years in China to improve cognition or as anti-aging
remedies. In our search for new ingredients from traditional Chinese medicinal
herbs, salidroside, a phenolic glycoside involved in cell anti-apoptosis
processes [5], was isolated from the rhizome of Rhodiola rosea L. (Crassulaceae).
However, the neuroprotective role of salidroside is unclear. The present study’s
aim was to explore whether salidroside could inhibit H2O2-induced toxicity in PC12 cells and the possible mechanism.
Materials and Methods
Materials
Salidroside was purchased from National Institute for the Control of
Pharmaceutical and Biological Products (Beijing, China). MTT, fluorescent
DNA-binding dye Hoechst 33258, and propidium iodide were purchased from
Sigma-Aldrich (St. Louis, USA). Dulbecco’s Modified Eagle’s Medium (DMEM) and
fetal bovine serum were obtained from Gibco Life Technologies (Grand Island,
USA). Lactic dehydrogenase (LDH) activity assay kit was obtained from
Jiancheng Institute of Biotechnology (Nanjing, China). Antibody of cytochrome c
was purchased from Santa Cruz Biotechnology (Santa Cruz, USA). DNA extraction
kit and caspase-3, -8 and -9 activity kits were from Beyotime Institute of
Biotechnology (Nantong, China). All other chemicals and reagents were of
analytical grade.
Cell culture and treatment and analysis of cell viability
Cells were cultured and treated as described by Qian et al
[6]. Briefly, PC12 cells were maintained in DMEM supplemented with
heat-inactivated 10% fetal bovine serum, 100 U/ml penicillin, 100 mg/ml
streptomycin in a water-saturated 5% CO2
atmosphere at 37 ºC. Experiments were carried out 48 h after cells were seeded
into 24-well plates. To produce oxidative stress, H2O2 was freshly prepared from 30% stock solution prior to each
experiment, and after 12 h exposure, the level of cellular MTT was quantified
as described by Chen et al [7]. Cells in 24-well plates were briefly
rinsed with phosphate-buffered saline (PBS), and 0.5 mg/ml MTT was added to
each well. The microplate was incubated at 37 ºC for an additional 4 h. At the
end of the incubation, the medium with MTT was removed and 500 ml dimethyl
sulfoxide was added to each well. The plate was shaken on a microplate shaker
to dissolve the blue MTT-formazan. The absorbance was read at 570 nm on a
microplate reader. When the effects of salidroside on the PC12 cells were
studied, different concentrations of salidroside were added simultaneous to
the medium just before the H2O2 was
added.
Measurement LDH release
LDH release was measured according to the method of Kruman et al
[8]. Cells were cultured in 24-well culture plates at a density of 1´104 cells/well for LDH assay.
After 12 h exposure to H2O2, LDH
activities in the medium were measured using an assay kit according to the
manufacturer’s instructions.
Hoechst staining
To quantify and assess nuclear morphology, PC12 cells were fixed for
10 min with 4% paraformaldehyde in PBS. The cells were then stained for 10 min
with 10 mg/ml fluorescent DNA-binding dye Hoechst 33258 to reveal nuclear
condensation [9]. Hoechst-stained cells were visualized and photographed under
a Leica DMIL microscope (Nussloch, Germany).
Analysis of DNA fragmentation
Fragmented DNA was isolated using a DNA extraction kit according to
the manufacturer’s instructions. The elutriants containing DNA pellets were
electrophoresed on a 1.8% agarose gel at 80 V for 1.5 h. The gel was examined
and photographed using an ultraviolet gel documentation system.
Flow cytometric analysis of DNA content
DNA content was measured according to the methods of Weinmann et
al [10]. Briefly, cells were collected and washed with ice-cold PBS and
fixed with 70% ethanol. The fixed cells were harvested by centrifugation at
1000 g for 5 min; dissolved in 100 ml PBS containing 50 mg/ml RNase A,
50 mg/ml propidium iodide, 0.1% Triton X-100 and 0.1 mM EDTA (pH 7.4); and then
incubated at 37 ºC for 30 min. The fluorescence of cell was measured by flow
cytometer (FACSCalibur; Becton Dickinson, San Jose, USA).
Reverse transcription-polymerase chain reaction (RT-PCR)
analysis
Total RNA was extracted from PC12 cells, and the potential residual
genomic DNA was eliminated with RNase-free-DNase I for 30 min at 37 ºC.
First-strand complementary DNA was synthesized as follows: 1 h at 42 ºC with
100 U Moloney murine leukemia virus reverse transcriptase (Promega, Madison,
USA), 15 U ribonuclease inhibitor (Promega), 500 mM dNTP, 0.5 mg oligo(dT)18
and 2 mg total RNA in a final volume 25 ml, and then 5 min at 95 ºC.
For PCR amplification, the specific primers included the control
glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 213 bp): 5¢-ATTCAACGGCACAGTCAAGG-3¢ (forward) and 3¢-AGTAGAGGCGGGGAAGACG-5¢ (reverse);
Bcl-2 (303 bp): 5¢-GATGACTTCTCTCGTCGCTA-3¢ (forward) and 3¢-TACGGAAACACCTTGATATA-5¢ (reverse); Bax
(331 bp): 5¢-GAACTGGACAATAATATGGA-3¢ (forward) and 3¢-TCACTGGTAGAAACACCGAC-5¢ (reverse). The
PCR mixture contained 0.8 pM forward and reverse primers of the Bax or Bcl-2,
0.4 pM forward and reverse primers of the GAPDH, 2.0 mM MgCl2, 200 mM deoxyribonucleotide triphosphate, and 1.5 U Taq DNA polymerase.
The PCR procedure was performed at 94 ºC for 5 min, followed by 28 cycles at
94 ºC for 1 min, 51 ºC for 30 s, 72 ºC for 45 s and extension at 72 ºC for 10
min. Next, 10 ml PCR products was mixed with 2 ml loading solution, and
electrophoresed on agarose-ethidium bromide gel at 100 V for 1 h. The gels were
examined and analyzed by an ultraviolet gel documentation system.
Analysis of caspase-3, -8, and -9 activities
Caspase-3, -8, and -9 activities were measured using assay kits
according to the manufacturer’s instructions. Supernatant was mixed with buffer
containing the recognition sequence for caspase attached to p-nitroanilide. The
absorbance of p-nitroanilide was determined at 405 nm. The caspase activities
were expressed as percentage compared to control.
Western blot analysis of cytochrome c
Cell lysates were prepared as described by Jia et al [11]. To
ensure equal loading of the protein samples, protein concentrations of the
cell lysates were determined by Bradford assay. Equal amounts of protein (30 mg in total) were
separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis
and transferred to a nitrocellulose membrane. The membrane was blocked with 5%
skim milk in 1´Tris-buffered saline containing 0.05%
Tween-20 (TBST) for 1 h. After blocking, the membrane was incubated with 1%
skim milk in TBST, containing the primary mouse monoclonal antibody against
cytochrome c (1:500) overnight. The membranes were then washed three
times with 1´TBST and then incubated with 1% skim
milk in TBST, containing a peroxidase-conjugated goat anti-mouse
immunoglobulin G secondary antibody (1:5000) (ZSGB-BIO, Beijing, China). The
detection of protein bands was performed using the 3,3¢-diaminobenzidine
tetrahydrochloride substrate kit (ZSGB-BIO).
Statistical analysis
All experiments were performed in triplicate. Data are presented as
mean±SD. The Duncan test and one-way ANOVA were used for multiple comparisons
using SPSS 12.0 software (SPSS, Chicago, USA).
Results
Inhibition of H2O2-induced
cytotoxicity
by salidroside
In PC12 cells, the protective effect on H2O2-induced cytotoxicity was assessed by MTT assay after 12 h incubation.
As shown in Table 1, when the cells were pre-incubated with salidroside
(10 and 100 mM), H2O2-induced cell toxicity
was significantly reduced in comparison with the control. Necrosis results in
a disruption of the cytoplasmic membrane, and the necrotic cells release
cytoplasmic LDH and other cytotoxic substances into the medium. We therefore
examined the existence of LDH in the cells’ culture medium. The LDH index was
significantly reduced at doses of 10 and 100 mM in comparison with the
control (Table 2). The results of MTT and LDH assays showed that
salidroside could have a protective effect against H2O2-induced cytotoxicity.
Salidroside
suppresses H2O2-induced apoptosis
Hoechst 33258 assay revealed the appearance of a collection of
multiple chromatin and fragmented apoptotic nuclei after treatment with 0.5 mM
H2O2 for 12 h. However, the apoptotic nuclei were significantly reduced
when cells were treated with 100 mM salidroside and 0.5 mM H2O2 [Fig. 1(A)]. After the PC12 cells were treated with 0.5 mM H2O2 for 12 h, DNA ladder pattern was detected, but salidroside was able
to reduce the ladder pattern in a dose-dependent manner [Fig. 1(B)]. When
the apoptotic cells were analyzed quantitatively by flow cytometry, a
significant increase in the apoptotic rate (from 9.78%±0.2% to 32.23%±4.0%) was
found after PC12 cells were treated with 0.5 mM H2O2 for 12 h. When PC12 cells were treated with 100 mM salidroside
and 0.5 mM H2O2 for 12 h, the percentage of apoptotic cells decreased from
32.23%±4.0% to 18.61%±1.5% [Fig. 1(C)].
Regulation of mRNA expression of Bax or Bcl-2 by
salidroside
As shown in Fig. 2, after H2O2 treatment for 6 h, mRNA expressions of Bax and Bcl-2
analyzed by RT-PCR analysis showed Bcl-2 expression began to decrease
and Bax expression began to increase. The effects of salidroside on
mRNA expression were investigated at the same indicated time. The results
showed salidroside (100 and 1 mM) significantly raised Bcl-2 expression and reduced Bax
in PC12 cells treated with 0.5 mM H2O2 (Fig.
2).
Salidroside
inhibits the activities of caspase-3, -8 and -9
To gain insight into the molecular effector pathway of H2O2-induced apoptosis, we first examined whether caspases were
downstream effectors in H2O2-mediated
apoptosis. H2O2 treatment caused a time-dependent increase in caspase-3, -8 and -9
proteolytic activities. However, when salidroside and H2O2 were added simultaneously to the medium, decreases in the activity
of caspase-3, -8 and -9 were detected (Fig. 3).
Salidroside
reduced cytochrome c in the cytosol
As indicated in Fig. 4, Western blot analysis revealed that H2O2 treatment caused a progressive accumulation of cytochrome c
in the cytosol. This was reduced when PC12 cells were treated with salidroside.
Discussion
Recently, researchers have made considerable efforts to search for
natural substances with neuroprotective potential, and particular attention has
been paid to Chinese medicinal plants with nootropic effect. The rhizome
of Rhodiola rosea L. has been used in East Asia as a tonic and
anti-aging agent since ancient times. There has been mounting evidence that the
extract from the rhizome of Rhodiola rosea L. possesses significant
neuroprotective activity and antioxidative effects [12,13], although little is
known about its pharmacological effects or active ingredients. In a previous
study, salidroside was isolated from the rhizome of Rhodiola rosea L. and
could significantly inhibit O2–– or H2O2-induced neurotoxicity in rat cortical cultures [14]. Earlier
results showed that 100 mM salidroside has little effect on PC12 cells, and there was no
significant difference compared with control group. The present findings
demonstrated that, in PC12 cells, salidroside reduced H2O2-induced apoptotic death caused by oxidative stress. Treatment with
salidroside significantly attenuated increased LDH leakage and decreased viability
in differentiated PC12 cells exposed to H2O2. In these
instances, the amount of H2O2 was greater than that of
salidroside, and the decrease in cell survival caused by H2O2 was nearly suppressed in the
presence of 0.1 mM salidroside. Therefore, we have speculated that
antioxidation is just one of salidroside’s pathways in this model. Inhibition
of relative targets in apoptosis might be a possible mechanism involved in the
protective effects of salidroside.
It has been well documented that some pathological neuronal
loss in AD occurs through apoptosis. The results of this present study showed
that salidroside protected PC12 cells against H2O2-induced apoptosis. Exposure to 0.5 mM H2O2 induced typical apoptosis in PC12 cells. These results were in accordance
with previous studies that found oxidative stress to be a common cause of
apoptosis [15,16]. When cells were pre-incubated with salidroside, H2O2-induced cell injury was significantly attenuated. For these
reasons, salidroside could be a useful neuroprotective agent to ameliorate
oxidative stress-induced apoptosis, which may be used in the treatment of AD.
Apoptosis is a type of cell death that represents the culmination
of naturally occurring or highly programmed mechanisms. Elucidating the expression
patterns of those factors during apoptotic cell death may be critical to our
understanding of the underlying mechanisms. Caspase-3 is a key executioner
caspase involved in neuronal apoptosis, and its activity is controlled by
upstream regulators, such as caspase-8 or caspase-9, which modulate the
mitochondria- and death receptor-dependent pathway, respectively [17]. The
present study showed that caspase-3 activity was up-regulated in H2O2-treated cells. We also detected enhanced caspase-9 activity in
H2O2-treated cells and the release of cytochrome c from
mitochondria into cytosol. Taken together, these results suggested that
H2O2-induced apoptosis in PC12 cells is associated with the
release of cytochrome c and the activation of caspases, probably
via the mitochondria-mediated apoptosis pathway. We further demonstrated
the down-regulation of Bcl-2 or up-regulation of Bax in H2O2-treated cells. Increased Bax and lowered Bcl-2 expression have been
shown to reduce mitochondrial membrane potential and increase reactive oxygen
species production in neurons [18], both of which are early events in the
process of apoptosis [19]. Our results suggested that the down-regulation of
Bcl-2 or up-regulation of Bax alters mitochondrial membrane permeability,
triggers mitochondrial cytochrome c release to cytosol and activates
caspase cascade.
Caspase-8 is a key initiating caspase involved in neuronal
apoptosis and that modulates the death receptor-dependent pathway. We detected
enhanced caspase-8 activity in H2O2-treated cells. The results suggested that the death
receptor-mediated pathway is involved in H2O2-induced apoptosis. However, recent studies have suggested that
caspase-8 is not always activated early in the context of Fas signaling. In
some cells, caspase-9 initiates the processing of caspase-3, which in turn
activates caspase-2 and -6. Caspase-6 was found to be required for the
activation of downstream caspase-8 [20]. In summary, our study suggested that
H2O2-induced apoptosis in PC12 cells is mediated by at least one pathway
through mitochondria that regulates the Bcl-2 family and caspase-3 and -9.
However, future studies are required to determine whether the death
receptor-mediated pathway is involved in H2O2-induced apoptosis.
Apoptosis is closely associated with the progression of AD and other
neurological diseases. In searching for anti-apoptosis agents, this study
examined the possible role of salidroside. Salidroside is an invaluable source
for the development of effective neuroprotective agents to protect against
apoptosis in PC12 cells in the treatment of age-related neurological diseases.
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