Salidroside Inhibits Endogenous Hydrogen Peroxide Induced Cytotoxicity of Endothelial Cells

Abstract

Salidroside, a phenylpropanoid glycoside isolated from Rhodiola rosea L., shows potent antioxidant property. Herein, we investigated the protective effects of salidroside against hydrogen peroxide (H₂O₂)-induced oxidative damage in human endothelial cells (EVC-304). EVC-304 cells were incubated in the presence or absence of low steady states of H₂O₂ (3–4 µM) generated by glucose oxidase (GOX) with or without salidroside. 3(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione (GSH) assays were performed, together with Hoechst 33258 staining and flow cytometric analysis using Annexin-V and propidium iodide (PI) label. The results indicated that salidroside pretreatment attenuated endogenous H₂O₂ induced apoptotic cell death in EVC-304 cells in a dose-dependent pattern. Furthermore, Western blot data revealed that salidroside inhibited activation of caspase-3, 9 and cleavage of poly(ADP-ribose) polymerase (PARP) induced by endogenous H₂O₂. It also decreased the expression of Bax and rescued the balance of pro- and anti-apoptotic proteins. All these results demonstrated that salidroside may present a potential therapy for oxidative stress in cardiovascular and cerebrovascular diseases.

Vascular endothelial cells serve as a barrier between tissue and blood stream, which plays an important role in maintaining vascular homeostasis. Oxidative stress has been implicated as a major cause of endothelial injuries in a variety of clinical abnormalities including artherosclerosis,¹⁾ ischemia reperfusion injury, and diabetes.^2,3)

Rhodiola rosea L. (Crassulaceae) has been used for a long time as folk medicine in Russia and China.⁴⁾ Its claimed benefits include anti-depress, anticancer, anti-stress, antioxidation and immune function improvement.⁵⁾ Salidroside (p-hydroxyphenethyl-β-D-glucoside, chemical structure shown in Fig. 1), a major active ingredient isolated from the plant R. rosea, has been used in the treatment of diabetes, hypertension, fatigue and hypoxia.^6,7) Recent study showed that salidroside had been found to exhibit marked antioxidant effects and its activity in scavenging superoxide radicals is concentration- and time-dependent.⁸⁾ Salidroside was also reported to be capable of protecting SH-SY5Y cells against hydrogen peroxide-induced cell apoptosis in a dose-dependent manner⁹⁾ and protecting the PC12 cells against hypoglycemia/serum limitation-induced cytotoxicity.¹⁰⁾

Fig. 1. Salidroside Inhibition of Endogenous H₂O₂-Induced Cell Damage

Cells were incubated with FBS-free medium, 10 mU GOX, different concentration of salidroside, or 10 mU GOX combined with different concentration of salidroside for 24 h. Cell proliferation was determined by MTT assay. Values are means±S.D. of five experiments. * p<0.05 compared with 10 mU GOX group and ^# p<0.05 vs. untreated cells group.

Although salidroside has been known to have significant anti-oxidative and neuroprotective properties, there are no reports on its effects on the vascular endothelial cells.

This study was to investigate whether salidroside could inhibit endogenous H₂O₂-induced toxicity in endothelial cells and the possible mechanism for protection.

MATERIALS AND METHODS

Chemicals and Reagents

Salidroside was provided by Tianjin Jianfeng Natural Product R&D Co., Ltd. (Tianjin, China), and the content of salidroside was about 2.0%, and the purity was above 98% (HPLC).

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Amresco Chemical Co., Ltd. EVC-304 cells were obtained from the Jilin Medical University. Dulbecco’s modified Eagle’s medium (DMEM) high glucose Medium was purchased from GIBCO BRL (Grand Island, NY, U.S.A.). Fetal bovine serum (FBS) was purchased from Hangzhou Sijiqing Co., Ltd. (Hangzhou, China). A Hoechst 33258 kit was purchased from Keygen of Nanjing PharMingen. Glucose oxidase (GOX, type X-S derived from Aspergillusniger, 100000–250000 U/g solid) was purchased from Sigma. Rabbit anti-human Bcl-2, Bax, pro Caspase-3, poly(ADP-ribose) polymerase (PARP), pro Caspase-9 and β-Actin Antibody were purchased from Epitomics Co., Ltd. (U.S.A.). Superoxide dismutase (SOD), malondialdehyde (MDA) and glutathione peroxidase (GSH-Px) assay kits were purchased from the Institute of Jiancheng Biology Engineering (Nanjing, Jiangsu, China).

Cell Culture

EVC-304 cells were grown in DMEM low glucose, supplemented with 10% fetal calf serum. The culture maintained at 37°C in a humidified atmosphere of 95% air and 5% CO₂. For experiments, cells were plated in 60 mm dishes and reached confluence within 2 to 3 d.

MTT Assay

EVC-304 cells (ca. 1×10⁴) were seeded in 96 well cell culture plates. After treatment with 10 mU GOX, different concentration salidroside, or 10 mU GOX cultured with different concentration salidroside for 24 h, and 20 µL MTT (5 mg/mL) were added to the media and incubated for 4 h at 37°C. The MTT solution was replaced by 150 µL dimethyl sulfoxide (DMSO) and agitated for 10 min. The absorbance was then read at 570 nm using an EL×800 Universal Microplate Reader (BIO-TEK, Norcross, GA, U.S.A.).

Measurement of MDA, Total SOD, and GSH-Px

Cells were incubated with FBS-free supplemented medium or medium containing 10 mU GOX, 100 µM salidroside, or 10 mU GOX cultured with 100 µM salidroside. Malondialdehyde, SOD and GSH-Px activities were all determined using commercially available kits. Total superoxide dismutase (T-SOD) activity was determined through xanthine oxidase method. Samples were taken to detect absorbance at 550 nm with a spectrophotometer. The calculated results were expressed by U/mL nitrite unit. GSH-Px activity was measured by dithio-dinitrobenzoic acid method at the absorbance of 412 nm and results were expressed by µmol/L protein. MDA content was measured using thiobarbituric acid (TBA) method at absorbance of 532 nm; results were expressed by nmol/mL protein. Methods and procedures were performed with assay kits according to the manufacturer’s instructions, respectively.

Fluorescence Microscopy

Cells were incubated with FBS-free supplemented medium or medium containing 10 mU GOX, 100 µM salidroside, or 10 mU GOX cultured with 100 µM salidroside. After 8 h incubation, cells were washed with phosphate buffered saline (PBS) twice, and then treated with the Hoechst 33258 kit. 3 h later, the cells were viewed under the fluorescence microscope (Nikon TE-2000U, Nikon Corporation, Tokyo, Japan).

Annexin-V–Propidium Iodide (PI) Assay

Cells were incubated with FBS-free supplemented medium or medium containing 10 mU GOX, 100 µM salidroside, or 10 mU GOX cultured with 100 µM salidroside. After 8h incubation, the media was then removed and cells were washed twice in ice cold PBS. Cells were suspended in 1×Annexin V binding buffer. Cells were transferred to a culture tube, and Annexin-V and PI were added; after gentle vortex, the cells were incubated for 15 min at room temperature in the dark. After adding 350 mL assay buffer to each tube, the suspension of each group was analyzed by FCM (BD FAC ScantoTM, U.S.A.).

Western Blot Analysis

Cells were seeded in 6-well plate. After treatment for 8 h, the cells were harvested and lysed in RIPA buffer (150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 50 mM Tris–HCl pH 7.4) for 30 min on ice. After centrifugation (13000×g 4°C, 15 min), the supernatant was loaded on to 12% polyacrylamide SDS gel. Each lane was loaded with 60 µg of cell lysate protein. After electrophoresis, the gels were blotted onto a polyvinylidene difluoride (PVDF) membrane, blocked with 5% (w/v) milk for 1 h on a shaker at room temperature, washed third with TBS-T for 10 min each, and then incubated with primary antibody at one of the following: Bcl-2, Bax, PRAP, pro caspase-3, pro caspase-9 at a dilution of 1 : 1000 overnight at 4°C. Primary antibody was detected with anti-rabbit immunoglobulin G (IgG) conjugated to horse-radish peroxidase (HRP), antibody dilutions were 1 : 5000, and visualized using ECL enhanced chemiluminescence.

Statistical Analysis

Data was represented as means±S.D. for at least three independent experiments. The differences between treatment groups were assessed by one-way ANOVA followed by unpaired Student’s t-test. Statistical significance was defined as p<0.05 to reject a null hypothesis.

RESULTS

Effects of Salidroside on EVC-304 Cells Exposed to Endogenous H₂O₂

Glucose oxidase catalyzes the oxidation of D-glucose in the presence of oxygen to D-gluconic acid and H₂O₂. The initial rates of H₂O₂ production by 10 mU GOX in HBSS was 1.5 µM H₂O₂/min.¹¹⁾ Under culture conditions, extracellular hydrogen peroxide (H₂O₂) concentration was maintained at approximately 7 to 9 mM with 10 mU GOX.¹⁾ In order to measure the effects of salidroside on endothelial damage induced by H₂O₂, the MTT reduction assay was used. As show in Fig. 1, 10 mU GOX induced significant decrease of cell survival when compared to control cells. In contrast, different concentration salidroside did not cause any apparent cyotoxicity, while the cells treated with 100 µM and 200 µM salidroside did not show visible changes in cell number (Fig. 1), indicating that salidroside protects against H₂O₂-induced cell damage.

Salidroside Represses MDA Production, and Restores the Activities of Total SOD and GSH-Px in EVC-304 Cells

Under disease conditions, oxygen free radical is the most lively and damaging free radical which is produced through enzyme and non-enzyme system. It may promote lipid peroxidation of polyunsaturated fatty acids (PUFA) of biomembranes, leading to formation of lipid peroxidessuch as MDA and hydroxyl, therefore resulting in tissue or cells damage. The elimination of free radical is dependent on the preventive or interrupted regulations of antioxidant defense system. SOD serves as an important member of the antioxidant system, which removes superoxides and protects cellsfrom damage. GSH-Px protects the structural integrity of cell membrane and function. Therefore the changes of MDA content, SOD and GSH-Px activity not only reflect the ability of scavenging oxygen free radical, but also indirectly reflect the extents of cell membrane damage attacked by free radical. The effects of salidroside on lipid peroxidation, and endogenous antioxidant preservation were evaluated as described in Materials and Methods. As shown in Fig. 2B, GOX alone increased MDA equivalents (thiobarbituric acid-reactive substances) in the control groups (p<0.01). In contrast, 100 µM salidroside significantly reduced MDA equivalents.

Fig. 2. Effects of Salidroside on the Protein Expression of SOD (A), MDA (B) GSH-Px (C), in EVC-304 Cells

Cells were incubated with 10 mU GOX, 100 µM salidroside or 10 mU GOX combined with 100 µM salidroside for 24 h. Results were expressed as means±S.D. (n=3). ^# p<0.05, compared to the untreated cells; ** p<0.01 vs. cells treated with 10 mU GOX alone.

Additionally, treatment with GOX alone decreased the activities of total SOD and GSH-Px, whereas treatment with 100 µM salidroside markedly attenuated the changes in total SOD (Fig. 2A) and GSH-Px (Fig. 2C) activities.

Salidroside Inhibited Endogenous H₂O₂-Induced Cell Apoptosis

Hoechst 33258 is a cell permeable blue fluorescent DNA dye to detect nuclear condensation, fragmentation and characteristics of apoptosis. As shown in Fig. 3A, apoptotic bodies containing nuclear fragments were significantly generated in 10 mU GOX-treated cells (Fig. 3A). However, treatment of 100 µM salidroside reduced cell apoptosis (Fig. 3A). Cells showed a normal Hoechst staining similar to that of control cells. To confirm this result, we stained the cells with Annexin-V, an early apoptotic marker, and PI for detection of late apoptosis. Flow cytometry was used to quantify fluorescent cells, and the results showed that salidroside significantly reduced the percentage of apoptotic cells (Figs. 3B, C).

Fig. 3. Salidroside Inhibition of Endogenous H₂O₂-Induced Cell Apoptosis

Cells were incubated with FBS-free medium, 10 mU GOX, 100 µM salidroside, or 10 mU GOX combined with 100 µM salidroside as indicated. A: Fluorescence microscopy examination of nuclear staining with Hoechst 33258; B: Flow cytometric analysis of Annexin-V stained cells; C: Quantitative analysis of data in B. Statistical significance indicated by ** p<0.01 vs. 10 mU GOX add with 100 µM salidroside.

Effect of Salidroside on Expression of Bcl-2 Family Proteins in Endogenous H₂O₂ Treated Cells

Members of the Bcl-2 family proteins such as Bcl-2 and Bax are critical regulators of the apoptotic pathway.¹²⁾ We examined the effect of salidroside on the expression of antiapoptotic Bcl-2 protein and pro-apoptotic Bax protein. As shown in Fig. 4, the protein level of pro-apoptotic protein, Bax, was declined, at the same time, the protein level of anti-apoptotic protein, Bcl-2, was increased in the cells treated with both 10 mU GOX and 100 µM salidroside when compared with 10 mU GOX group.

Fig. 4. Western Blot Analysis of Bcl-2 and Bax Expression

A: Total cell extracts were analyzed with western blotting using a rabbit polyclonal antibody to Bcl-2, Bax and β-actin levels were performed to assess the total amount of proteins loaded on the gel; a. Untreated cells; b. 10 mU GOX; c. 100 µM salidroside; d. 10 mU GOX add 100 µM salidroside. B: Data obtained from quantitative densitometry was presented as mean±S.D. of 3 independent experiments. * p<0.05 vs. 10 mU GOX.

Effect of Salidroside on Expression of Caspase Protease Family in Endogenous H₂O₂ Treated Cells

Caspase, a family of cysteine proteases, is known to form integral parts of the apoptotic pathway¹³⁾. Therefore, we investigated the protein levels and activities of caspase-3. As shown in Fig. 5, the cleavage of precursor of caspase-9 and caspase-3 was noted in GOX treated cells. PARP, an enzyme involved in DNA repair, is a substrate for caspase-3.¹⁴⁾ Ten milliunit GOX treatment caused cleavage of PARP, 116 kDa into 89 kDa fragment (Fig. 5). However, the excessive activation of caspase-3 and cleavage of PARP were ameliorated in the salidroside treatment groups. The action of salidroside on these molecular changes was paralleled with its effects on apoptosis.

Fig. 5. Western Blot Analysis of Caspase-3,9 and PARP Expression

A: Total cell extracts were analyzed with Western blotting using a rabbit polyclonal antibody to Caspase-3,9 and PARP, and β-actin levels were performed to assess the total amount of proteins loaded on the gel. a. Untreated cells; b. 10 mU GOX; c. 100 µM salidroside; d. 10 mU GOX add 100 µM salidroside. B: Data obtained from quantitative densitometry was presented as mean±S.D. of 3 independent experiments. * p<0.05 vs. 10 mU GOX.

DISCUSSION

It has been well established that the release of reactive oxygen species (ROS) from vessel tissue was elevated under pathological conditions. ROS, such as hydrogen peroxide, superoxide anion and hydroxyl radicals, readily damage biological molecules, which could ultimately lead to apoptosis or cell death.^15,16) Thus, protection of endothelial cells from ROS-induced injury may therefore provide beneficial therapeutical intervention for successful treatment of cardiovascular diseases.¹⁷⁾ Salidroside, a major active ingredient exsiting naturally in R. rosea L., has been reported to be a strong antioxidant. In the present study, we demonstrated for the first time that salidroside was capable of protecting EVC-304 cells from oxidative stress and the accompanied apoptosis caused by endogenous H₂O₂. Then we further explored the possible molecular mechanisms underlying the antioxidative effects of salidroside on protecting EVC-304 cells against endogenous H₂O₂-induced cell apoptosis.

H₂O₂ is continuously produced in vivo and remains in a quasi steady state: its concentration changes in a time scale slower than its turnover.¹⁾ Hence, exposing cells to steady state concentrations of H₂O₂, instead of bolus additions, constitutes a superior method for oxidant delivery that mimics the physiological settin.¹⁸⁾ Enzymatic generation of low and steady H₂O₂ fluxes may also be appropriate in cell culture studies, because the bolus addition of H₂O₂ may not adequately reflect its production or delivery in vivo.^11,19–21) In this regard, the GOX system may be useful in mimicking the mode of extracellular oxidant production from physiological sources such as xanthine oxidase and reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase.^22,23) Our present studies confirmed that treating cells with 10 mU GOX resulted in a cell viability loss. However, when incubated with 100 µM and 200 µM salidroside, the cell viability loss was greatly decreased. These results indicated that salidroside did significantly protect EVC-304 cells from H₂O₂-induced cytotoxicity.

H₂O₂ ultimately lead to apoptotic or necrotic cell death. We further explored whether salidroside has a protective effect against EVC-304 cell apoptosis induced by H₂O₂. 10 mU GOX treated cells stained with fluorescent DNA binding dye, Hoechst 33258, displayed typical morphological features of apoptosis with sickle shaped-nuclei. Salidroside could mitigate these morphological changes. To confirm this result, we stained the cells with Annexin-V, an early apoptotic marker, and PI for detection of late apoptosis. Flow cytometry was used to quantify fluorescent cells, and the result showed that salidroside significantly reduced the percentage of apoptotic cells. All these results signified that salidroside undoubtedly plays a key role in preventing cells from undergoing H₂O₂-induced apoptosis.

Membrane lipid is a major target for intracellular ROS.²⁴⁾ Lipid peroxidation, generally assessed by MDA formation, may disrupt the integrity of plasma membrane. On the contrary, endogenous antioxidases including SOD and GSH-Px can compromise the excessive ROS in vivo to regulate intracellular redox status.²⁵⁾ Therefore, abrogating occurrence of lipid peroxidation and increasing levels of endogenous antioxidases may provide a repairing mechanism for ROS-mediated cell damage. The present study suggests that 100 µM salidroside significantly increased SOD and GSH-Px activities in endogenous H₂O₂ treated EVC-304 cells, along with an attenuated MDA production. These results suggested that enhancement of endogenous antioxidant preservation and subsequent attenuation of lipid peroxidation may represent a primary mechanism of cellular protection for salidroside.

Mitochondria are thought to be a pathway for apoptosis and its function is regulated through Bcl-2 family proteins.²⁶⁾ To examine further mechanism of anti apoptosis of salidroside, we investigated the expression of some Bcl-2 family members including a wide variety of anti-apoptotic proteins as well as pro-apoptotic proteins, such as Bax. Bcl-2 inhibits apoptosis by negatively regulating the apoptotic activity of Bax and forming Bcl-2/Bax heterodimers. The Bcl-2/Bax ratio is a measure of the cell death switch, which determines whether a cell will live or die upon being exposed to an apoptotic stimulus. Thus, the expression of the Bcl-2 and Bax proteins in the cells of each group were measured using western blots. Our current study demonstrated a remarkable increase of the Bax and decrease of Bcl-2 at protein levels after salidroside treatment.

PARP is the substrate for effective caspases during apoptosis, which is involved in DNA repair, genome surveillance, and maintenance of genomic integrity in response to environmental stress. The cleavage of PARP is the hallmark of apoptosis.²⁷⁾ The result showed that caspase-mediated PARP cleavage induced by 10 mU GOX apoptosis was decreased by incubation with salidroside. We further examined the involvement of Caspases in H₂O₂-mediated apoptosis. Compared with the control group, caspases-3, 9-were inactivated after combination with salidroside.

In summary, salidroside could ameliorate H₂O₂ induced oxidative stress and apoptosis in EVC-304 cells. These data strongly support that salidroside plays a protective role in H₂O₂-induced endothelial injury as a potent antioxidant. Due to its efficacy, salidroside might be a potential therapy for oxidative stress in cardiovascular and cerebrovascular diseases.

Acknowledgements

This study was supported by a Grant from Science Research Fund of Department of Education of Jilin Province (No. 2008265, No. 2009311, No. 2012335, No. 2008051 and No. 2012334). Authors also acknowledge grant support from the following: National High-Tech R&D Program (863 Program) (Program Number: 2012AA021902), National Natural Science Foundation of China (Program Number: 81102367), the “Science and Technology Research and Development Project” of Shaanxi Province (Grant No. 2011KTCL03-23 and 2011K12-03-10), and Xi’an Science and Technology Bureau (Grant No. CXY1131).

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