|
|
|
Original Paper
|
|
|||
|
Acta Biochim Biophys
Sin 2008, 40: 796-802 |
||||
|
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 apop�tosis 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 neuro��dege�nerative 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 pathologi�cal 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 �C. 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 �C 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 �C for 30 min. The fluorescence of cell was measured by flow
cyto�meter (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 �C. First-strand complementary DNA was synthesized as follows: 1 h at 42 �C 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 �C. For PCR amplification, the specific primers included the control glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 213 bp): 5�-ATTCAACGGCA�CAGT�C�A�AGG-3� (forward) and 3�-AGTAGAGGCGG�GGAA�G�A�C�G-5� (reverse); Bcl-2 (303 bp): 5�-GATGACTTCTCTCGTCGC�T�A-3� (forward) and 3�-TACGGAAACA�CC�T�T�G�A�TATA-5� (reverse); Bax (331 bp): 5�-GAACTGGACAATAATATG�GA-3� (forward) and 3�-TCACTGGTA�G�A�A�A�CACCGAC-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 �C for 5 min, followed by 28 cycles at 94 �C for 1 min, 51 �C for 30 s, 72 �C for 45 s and extension� at 72 �C 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 poly�acrylamide 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�-diamino�benzidine
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 cyto�plasmic 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.
References
1�� Lassmann H, Bancher C, Breitschopf H, Wegiel
J, Bobinski M, Jellinger K, Wisniewski HM. Cell death in Alzheimer�s disease evaluated
by DNA fragmentation in situ. Acta Neuropathol 1995, 89: 3541
2�� Smale G, Nichols NR, Brady DR, Finch CE,
Horton WE Jr. Evidence� for apoptotic cell death in Alzheimer�s disease. Exp
Neurol 1995, 133: 225-230
3�� Guan S, Bao YM, Jiang B, An LJ. Protective
effect of protocatechuic acid from Alpinia oxyphylla on hydrogen
peroxide-induced oxidative PC12 cell death. Eur J Pharmacol 2006, 538: 73-79
4�� Tang XQ, Feng JQ, Chen J, Chen PX, Zhi JL,
Cui Y, Guo RX et al. Protection of oxidative preconditioning against
apoptosis induced by H2O2 in PC12 cells:
mechanisms via MMP, ROS, and Bcl-2. Brain Res 2005, 1057: 57-64
5�� Zhang WS, Zhu LQ, Deng RC, Niu FL, Tian R.
Effect of salidroside on mitochondrial membrane potential during injury induced
by hypoxia/hypoglycemia in cultured SH-SY5Y cells. Chinese Journal of
Pathophysiology 2004, 20: 1218-1221
6�� Qian YF, Wang H, Yao WB, Gao XD. The aqueous
extract of the Chinese medicine Danggui-Shaoyao-San inhibits apoptosis in
hydrogen peroxide-induced PC12 cells by preventing cytochrome c release
and inactivating of caspase cascade. Cell Biol Int 2008, 32: 304-311
7�� Chen P, Li A, Zhang MJ, He ML, Chen Z, Wu XK,
Zhao CJ et al. Protective effects of a new metalloporphyrin on
paraquat-induced oxidative stress and apoptosis in N27 cells. Acta Biochim
Biophys Sin 2008, 40: 125-132
8�� Wang H, Yao WB, Qian YF, Gao XD. Protective
effect of salidroside on apoptosis in PC12 cells induced by cyanide and glucose
deprivation. Journal of China Pharmaceutical University 2007, 38: 273-276
9�� Kruman I, Bruce-Keller A, Bredesen D, Waeg G,
Mattson MP. Evidence that 4-hydroxynonenal mediates oxidative stress-induced
neuronal apoptosis. J Neurosci 1997, 17: 5089-5100
10� Weinmann P, Scharffetter-Kochanek K, Forlow
SB, Peters T, Walzog B. A role for apoptosis in the control of neutrophil
homeostasis in the circulation: insights from CD18-deficient mice. Blood 2003,
101: 739-746
11� Jia RR, Gou YL, Ho LS, Ng CP, Tan NH, Chan HC.
Anti-apoptotic activity of Bak Foong Pills and its ingredients on 6-hydroxydopamine-induced
neurotoxicity in PC12 cells. Cell Biol Int 2005, 29: 835-842
12� Xu Q, Guo ZY, Kang JS, Zhu SG, Du KQ, Yang HF.
Study on protection of Rhodiola sachalinensis AR against free radical
injury of the ischemia reperfusion rats. Journal of Mormon Bethune University
of Medical Sciences 1999, 25: 232-234
13� Lee MW, Lee YA, Park HM, Toh SH, Lee EJ, Jang
HD, Kim YH. Antioxidative phenolic compounds from the roots of Rhodiola
sachalinensis A. Bor. Arch Pharm Res 2000, 23: 455-458
14 Li TW, Kong LK, Mu JY, Li XM, Yang HY. The
protection of salidrosides against O2-
or H2O2 damage to rat cortical
cultures. China Academic Journal Electronic Publishing House 1997, 14: 143-144
15� Whittemore ER, Loo DT, Watt JA, Cotman CW. A detailed
analysis of hydrogen peroxide-induced cell death in primary neuronal culture.
Neuroscience 1995, 67: 921-932
16� Satoh T, Sakai N, Enokido Y, Uchiyama Y,
Hatanaka H. Survival factor-insensitive generation of reactive oxygen species
induced by serum deprivation in neuronal cells. Brain Res 1996, 733: 9-14
17� Kuida K, Zheng TS, Na S, Kuan C, Yang D,
Karasuyama H, Rakic P et al. Decreased apoptosis in the brain and
premature lethality in CPP32-deficient mice. Nature 1996, 384: 368-372
18� Pastorino JG, Chen ST, Tafani M, Snyder JW,
Farber JL. The overexpression of Bax produces cell death upon induction of the
mitochondrial permeability transition. J Biol Chem 1998, 273: 7770-7775
19� Marchetti P, Hirsch T, Zamzami N, Castedo M,
Decaudin D, Susin SA, Masse B et al. Mitochondrial permeability
transition triggers lymphocyte apoptosis. J Immunol 1996, 157: 4830-4836
20 Slee EA, Harte MT, Kluck RM, Wolf BB, Casiano
CA, Newmeyer DD, Wang HG et al. Ordering the cytochrome c-initiated
caspase cascade: hierarchical activation of caspases-2, -3, -6, -7, -8, and -10
in a caspase-9-dependent manner. J Cell Biol 1999, 144: 281-292