Shenfu injection protects human ECV304 cells from hydrogen peroxide via its anti-apoptosis way

Journal of Ethnopharmacology 163 (2015) 203–209

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Shenfu injection protects human ECV304 cells from hydrogen peroxide via its anti-apoptosis way Hong Fen-fang a,b,1, Guo Fa-xian a,1, Zhou Ying c,1, Min Qin-hua d,1, Zhang Da-lei a, Yang Bei a, Zhou Wei-ying a, Wu Lei a, Wei Zhi-ping a, Liu Hui a, Yang Shu-long a,n a

Department of Physiology, College of Medicine, Nanchang University, Nanchang 310006, China Department of Experimental Teaching, College of Medicine, Nanchang University, Nanchang 310006, China c Department of Tissue Embryology of Basic Medical College, Nanchang 310006, China d The First Affiliated Hospital of Obstetrics and Gynecology, Nanchang 310006, China b

art ic l e i nf o

a b s t r a c t

Article history: Received 5 September 2014 Received in revised form 20 January 2015 Accepted 24 January 2015 Available online 4 February 2015

Ethnopharmacological relevance: The pathogenesis of thromboangiitis obliterans (TAO) has not been fully elucidated until now. Shenfu injection (SFI), a traditional Chinese formula has been widely used clinically for the treatment of cardiovascular diseases for more than two decade. Our previous results first suggested that SFI can cause a significant therapeutic effect on experimental TAO model rats. This experiment was designed to further investigate the protective effect of SFI on VEC damaged by hydrogen peroxide (H2O2) oxidative stress in vitro. Meterials and methods: The cell viability was evaluated by the MTT assay, the activities of SOD and GSH-PX and the content of MDA in the supernatants of the cultured ECV304 cells were evaluated by a colorimetry method, cell apoptosis was detected by flow cytometry and an AO/EB double staining method. The protein expressions of Bcl2, Bax and caspase-3 were examined by Western blotting. Results: When compared with control group, lower survival rate of ECV304 cells was observed in H2O2 group (po 0.01) ; 20 μl/ml, 30 μl/ml and 40 μl/ml SFI increased the survival rate of ECV304 cells under H2O2 oxidative stress (p o0.05 and po 0.01). The activities of SOD and GSH-PX were higher and MDA level was lower in H2O2 group than those in control group. These effects of H2O2 on SOD, GSH-PX activities and MDA content were reversed by SFI in concentration-dependent way (po0.05 and po 0.01). Flow cytometry and AO–EB double staining discovered that SFI pretreatment inhibited the ECV304 cells apoptosis. The protein expression of caspase3 in 30 μl/ml and 40 μl/ml SFI groups significantly decreased whereas Bcl2 protein expressions in 20 μl/ml, 30 μl/ml and 40 μl/ml SFI groups were higher than H2O2 group, with Bax protein expression much lower than H2O2 group (p o0.05 and po0.01). Conclusions: Our findings suggest that SFI could prevent the ECV304 cells against H2O2 oxidative-stress by enhancing antioxidant enzyme activities, reducing the membrane lipid peroxidation, as well as upregulating antiapoptotic and downregulating apoptosis protein expressions. & 2015 Elsevier Ireland Ltd. All rights reserved.

Keywords: Shenfu injection Vascular endothelial cell H2O2 Anti-oxidation Apoptosis

1. Introduction Thromboangiitis obliterans (TAO) is a chronic nonatherosclerotic segmental occlusive vasculitis with arterial lumen thrombosis, which most commonly involves the small- and medium-sized arteries and veins of the extremities and usually leads to gangrene and tissue loss (Hong et al., 2011; Dargon and Landry, 2012; Weinberg and Jaff, 2012; Vijayakumar et al., 2013). At present, TAO accounted for 40–60% of peripheral vascular diseases in eastern parts of the world. However, since the disease was first descripted by Winiwarter almost

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Corresponding author. Tel: þ 86 791 86360556. E-mail address: [email protected] (Y. Shu-long). 1 Co-first authors. http://dx.doi.org/10.1016/j.jep.2015.01.032 0378-8741/& 2015 Elsevier Ireland Ltd. All rights reserved.

130 years ago, the pathogenesis of TAO has not been fully elucidated (Malecki et al., 2009). Evidences have indicated a close relationship existed between TAO and damaged vascular endothelial cells (VEC). TAO patients have an intrinsic decrease in endothelial progenitor cells not entirely associated with smoking, which may be the cause of endothelial dysfunction seen in TAO patients leading to the development of this disease at early ages (Park et al., 2013). Fazeli et al. have proved that Sera from TAO patients could activate human umbilical vein endothelial cells (HUVECs) and suggested that this same activation might occur in vivo by the responsible cytokines, in particular those released from activated platelets, free oxygen radicals, and possibly low levels of NO of the sera of TAO patients, as a consequences of chronic cigarette smoking and of endothelial NO synthase polymorphism (Fazeli et al., 2014). Recently, cigarette smoking extract (CSE) which

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is an important risk factor in the onset and development of TAO, has been proved to injury the function of HUVECs and Atorvastatin can protect endothelial function against CSE, the underling mechanism may be related to cell morphology's change, the expressions of eNOS and the release of NO as well as the protein expressions of ICAM-1 (Xu et al., 2011). Excessive oxidation of VEC induced by hydrogen peroxide(H2O2) is one of the main factors resulting in its apoptosis, in which the morphology and function of VEC were damaged (Fujita et al., 2000; Ni et al., 2013; Sun et al., 2013). Excessive oxidation of VEC is also one of the most important reasons to promote intravascular thrombosis of TAO (Ming-Chao et al., 2008; Park et al., 2013). Furthermore, thrombosis is believed to play a prominent role in the pathogenesis of TAO (Gaffo, 2013). For example, by establishing in vitro TAO model of an HUVECs cell line ECV304 with H2O2 incubation, Cui Leisun et al. revealed that Jiajianqingying Recipe, a famous traditional Chinese formulation comosed of Salvia miltiorrhiza, Radix paeoniae rubra, Radix rehmanniae, Honeysuckle, forsythia, and Pubescent Holly root, etc, could obviously protect ECV304 from H2O2 oxidation, suggesting that Jiajianqingying Recipe is the effective agent to control TAO (Ming-Chao et al., 2008). Until now there has been no effective remedy to cure TAO fundamentally, the poorly clinical treatment for the disease just aims at alleviating the pain of this patients (Dargon and Landry, 2012; Vijayakumar et al., 2013). TAO belongs to “gangrene” category of traditional Chinese medicine. It can be traced back to Huangdi Neijing published in the spring and autumn period in ancient China. Increasing data shows that the traditional Chinese medicine has its unique advantage in treatment of TAO (Zhang et al., 2001; Guo, Yang 2013). Shenfu injection (SFI) originated from Shenfu Tang, which is a traditional Chinese formulation curing the patients from collapse by restoring Yang. And SFI is mainly composed of the extract of radix ginseng and radix aconitum carmichaeli root (Hong et al., 2011). Up to now, SFI has been widely used clinically for the treatment of cardiovascular diseases for more than two decade. SFI has the potential to exert cardioprotective effects against Fupian injection toxicity, which possibly correlated with the activation of cytochrome P450 2J3 (Xiao et al., 2013). In addition, SFI also exhibits neuroprotective effects for neonatal hypoxic-ischemic brain injury by preventing neuron apoptosis and has potential to be used in the clinical for the treatment of perinatal hypoxia–ischemia (Ni et al., 2013). Recently, SFI is shown to cause an apparent thoracic aorta relaxation by endothelium-dependent manner (Zhu et al., 2013).Our previous results first suggested that SFI can cause a significant therapeutic effect on experimental TAO model rats by its inhibiting platelet aggregation and enhancing anti-thrombus function of vessel endothelia (Hong et al., 2011). In this study, the human endothelial cell line ECV304 was used to make a VEC injury model induced by H2O2 for further investigating the mechanism underlie the protective effect of SFI on the damaged VEC in vitro.

2. Materials and methods

Antibodies for Bax, Bcl-2, caspase-3, β-actin and horseradish peroxidase (HRP)-conjugated secondary goat anti-rabbit/anti-rat antibody were from Santa Cruz (California, USA). Annexin V-FITC apoptosis detection kit was purchased from Nanjing Vazyme Biotech Co., Ltd. (Nanjing, China). Hydrogen peroxide (H2O2) was purchased from Xilong Chemical Co., Ltd. (Shantou, China). Malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) assay kits were supplied by Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Bicinchoninic acid assay (BCA) kit and radioimmunoprecipitation assay (RIPA) lysis buffer were purchased from the Beyotime Institute of Biotechnology (Shanghai, China). All other chemicals were of the highest purity commercially available. H2O2 was freshly prepared for each experiment from a 30% stock solution.

2.2. Cell culture and drug treatments Human umbilical vein endothelial cell line ECV304 was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and maintained in M199 medium containing 10% fetal bovine serum (FBS), L-glutamine (2 mM), 100 U/mL of penicillin, and 100 μg/ mL streptomycin at 37 1C in a humidified atmosphere of 5% CO2. The cell passage was within 10 passages from when the cell line was purchased. For all experiments, ECV304 cells were grown to 70–80% confluence and then pretreated with designated concentrations of SFI for 3 h prior to H2O2 exposure in fresh medium. SFI was diluted by Phosphate buffer, and Phosphate Buffered Saline (PBS) was used as the control in all experiments.

2.3. Cell viability assay Cell viability was assessed by a modified MTT assay. Cells (1  105 cells/mL) were seeded to 96-well plates and incubated for 24 h.Then, the cells were exposed to SFI at various concentrations for 3 h prior to 3 h treatment with 300 μM H2O2. Thereafter, 10 μL of 5 mg/mL MTT in phosphate buffered saline (PBS) was added to each well and cells were incubated for a further 4 h at 37 1C. Then, the medium was then replaced by 100 μL of DMSO to dissolve the formed precipitate. The optical density was measured in a microplate reader (Model 680, Bio-Rad, USA) at 490 nm.

2.4. Evaluation of MDA levels, and SOD and GSH-PX activities ECV304 cells were pretreated with SFI at different concentrations for 3 h, followed by exposure to 300 μmol/L H2O2 for an another 3 h. Then ECV304 cells were harvested with 0.25% trypsin and washed twice with PBS. The cells were lysed with lysis buffer and centrifuged at 4000 rpm at 4 1C for 10 min. MDA levels and the activities of SOD and GSH-Px in the supernatant were measured spectrophotometrically using detection kits according to manufacturer instruction, respectively.

2.1. Materials 2.5. Flow cytometric analysis of apoptotic cells Shenfu injection (SFI) originated from the traditional Chinese formula of Shenfu Tang, and its main components are ginsenoside and aconitum alkaloid. It was purchased from Yaan Sanjiu Pharmaceutical Co., Ltd. (Sichuan, China), 10 mL/piece, per milliliter of SFI included 0.5 mg of ginsenoside and 0.1 mg of aconitine. 3-(4,5dimethylthiazol-2-yl)- 2,5-dephenyltetrazolium bromide (MTT) was obtained from Sigma-Aldrich (St. Louis, Misouri, USA). Acridine orange (AO) and ethidium bromide (EB) were from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Dulbecco's modified Eagle's medium (DMEM), L-glutamine, penicillin, and streptomycin were purchased from Gibco BRL (Grand Island, New York, USA).

In order to evaluate the apoptotic rate of ECV304 cells, doublestaining assay was performed using an annexin V-FITC/propidium iodide (PI) kit. ECV304 cells were treated with various concentrations of SFI for 3 h prior to exposure to 300 μmol/L H2O2 for 3 h. At the end of the treatment, cells were collected and washed with PBS twice, and then resuspended in binding buffer at a concentration of 1  106 cells/mL. Then, 5 μL of annexin V-FITC and 5 μL of PI were added. The cells were incubated for 15 min in the dark. After staining, the quantification of apoptotic cells was analyzed by flow cytometry.

H. Fen-fang et al. / Journal of Ethnopharmacology 163 (2015) 203–209

2.7. Western blot analysis Cells were pre-treated with various concentrations of SFI for 3 h in the absence or presence of H2O2. Proteins from every treatment were extracted in lysis buffer. After lysis on ice for 30 min, cell lysates were clarified by centrifugation at 12,000 rpm at 4 1C for 10 min. Protein concentrations were determined by a BCA. Immunoblot analysis of protein expressions of Bax, Bcl2, caspase3 was performed as described in our previous report. Briefly, 50 μg equal amounts of protein extracts were separated by 12% SDS–polyacrylamide gels, then proteins were transferred onto PVDF membranes that were from Millipore (Massachusetts, USA). The membranes were blocked in 5% nonfat milk powder in Tris-buffered saline/0.1% Tween-20 (TBST) for 1 h at room temperature, and then incubated overnight with the primary antibody at 4 1C and horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature. After washing thrice, the immunoblots were detected by enhanced chemiluminescence (ECL) detection reagent (TianGen Biotech Co., Ltd., Beijing, China). Relative band intensity of each protein was normalized for b-actin.

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Morphological assessment of apoptotic cells was performed using the AO/EB double staining method. ECV304 cells were seeded in 24-well microtiter plates (10,000 cells per well). After 24 h incubation, cells were firstly treated with SFI (20,30 or 40 μl/ml) for 3 h, then incubated with 300 μmol/L H2O2 for another 3 h at 37 1C in a 5% CO2 atmosphere. Afterwards, AO/EB double dye solution (AO and EB100 Ug/m1) 4 ml mixing solution was added each well in microtiter plates. The morphology of the cells was observed and photographed with a fluorescence light microscope ((Nikon Eclipse E800 microscope). Viable cells stained only by AO were bright green with intact structure; early apoptotic cells stained by AO were bright green area in the nucleus. Late apoptotic cells stained by AO and EB were red– orange with condensation of chromatin as dense orange areas and reduced cells size seen in this study (Wang and Huang, 2005; Yang et al., 2006).

lower concentrations of SFI (e.g.,5, 10, 20, 30, 40 μl/mL) did not affect the viability of ECV304 cells, whereas concentrations higher than 80 μl/mL reduced cell viability (po0.05) (Fig. 1B). Hence, 20, 30, and 40 μl/mL of SFI were used for the following experiments. Furthermore, to evaluate any protective effect of SFI against H2O2-induced injury in ECV304 cells, ECV304 cells were incubated with SFI at different concentrations (20, 30, and 40 μl/mL) for 3 h prior to the exposure to 300 μM H2O2 for 3 h. After this experimental procedure, cell viability was measured by MTT assay indicated as Section 2. As shown in Fig. 1C, the decreased cell viability induced by H2O2 was significantly attenuated by SFI pretreatment in a concentrationdependent manner. These observations suggest that SFI protected ECV304 cells from H2O2-induced cell injury.

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To determine the appropriate concentration of H2O2 inducing oxidative stress in ECV304 cells, the cells were treated with various concentrations of H2O2 for 3 h. Cell viability was assessed by MTT assay and expressed as a percentage of control. As indicated in Fig. 1A, the concentration of H2O2(50,100,200,300,400,800 μM) incubated with ECV304 cells for 3 h damaged ECV304 cells in concentration-dependent manner. 300 μM H2O2 incubated with ECV304 cells for 3 h was shown to be a suitable condition to make an oxidative-stress damaged ECV304 cells model with about 50% cellular proliferation inhibition rate (po0.01). To determine adequate concentrations of SFI in the experiments, ECV304 cells were incubated with various concentrations of SFI for 3 h. As shown in Fig. 1B, the

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SFI concentration Fig. 1. Effects of H2O2(A) and SFI(B) on ECV304 cell viability and SFI action on the injured ECV304 cell viability by H2O2(C). ECV304 cells were treated with various concentrations of H2O2 (50, 100, 200, 300, 400, and 800 μM) (Fig. 1A) or SFI (5, 10, 20, 30, 40, 80, and 160 μl/ml) (Fig. 1B) for 3 h. In addition, cells were incubated with 20, 30 and 40 μl/mL of SFI for 3 h prior to the exposure to 300 μM H2O2 for 3 h (Fig. 1C). Cell viability was assessed by MTT assay and expressed as a percentage of control. Each point represents the mean 7S.D. of three replicate values in three different experiments. np o 0.05 and nnp o 0.01 compared to control; p o 0.05 and p o0.01 compared with the H2O2 treated cells.

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3.2. Effects of SFI on MDA level, the activities of total SOD and GSHPx in ECV304 cells As shown in Fig. 2, the treatment of ECV304 cells with 300 μM H2O2 for 3 h caused a significant decrease in the activities of total SOD and GSH-Px (po0.01) and an increase in MDA levels when compared to the control cells (po0.01). However, pretreatment of the cells with SFI (20, 30, and 40 μl/mL) markedly attenuated the decreased activities of SOD and GSH-Px, while it significantly reduced the increased MDA levels induced by H2O2, in a concentration-dependent manner. 3.3. Effects of SFI on H2O2-induced apoptosis in ECV304 cells by flow cytometry and AO/EB double staining In order to examine the protective effect of SFI against apoptosis induced by H2O2 in ECV304 cells, cell apoptosis rates in different

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groups were measured by the annexin V-FITC/PI double staining method with flow cytometry (Fig. 3A and B). In the scatter plot of double variable flow cytometry, Q3 quadrant (FITC /PI ) shows living cells; Q2 quadrant (FITCþ/PIþ ) stands for late apoptotic cells; and Q4 quadrant(FITCþ /PI ) represents early apoptotic cells. As displayed in Fig. 3A and B, the percentage of apoptotic cells was higher in the H2O2 group than that in the control group. But a marked dose-dependent decrease in both early and late stages of apoptosis was obvious in ECV304 cells after 20, 30, and 40 μl/mL SFI treatment when compared with H2O2 group. Under the fluorescence light microscope, the normal morphologies of ECV304 cells in control group appeared to be green nuclei and intact structure (Fig. 3C), when treated with 300 μmol/L H2O2 for 3 h, the ECV304 cells shown shrinkage, membrane blebbing, chromatin condensation, and formation of apoptotic bodies. However, the ECV304 cells pre-incubated with 20, 30, and 40 μl/mL SFI prior to H2O2-induced oxidative stress displayed slightly morphological changes of apoptosis by AO/EB double staining in a concentration-dependent manner. The results suggested that SFI protected ECV304 cells against H2O2-induced apoptosis. 3.4. Effects of SFI on protein expressions of Bcl-2, Bax and caspase-3

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In addition, we further determined the effects of SFI on the protein expressions of Bax, Bcl-2 and caspase-3 in H2O2-induced ECV304 cells by Western blot. As shown in Fig. 4, the treatment of ECV304 cells with 300 μM H2O2 significantly increased the protein expressions of Bax and caspase-3, while significantly decreased Bcl-2 levels, when compared with the control cells (po0.01). However, pretreatment with SFI at concentrations of 20, 30, and 40 μl/mL remarkably enhanced Bcl-2 expression and inhibited Bax and caspase-3 expression in a concentration dependent manner.

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SFI concentration Fig. 2. Effects of SFI on (A) superoxide dismutase (SOD) activity, (B) GSH-Px activity, and (C) malondialdehyde (MDA) production in H2O2-induced ECV304 cells. SFI significantly decreased the level of MDA, while increasing the activities of SOD and GSH-Px in a concentration-dependant manner in H2O2-induced ECV304 cells. Data are presented as the mean 7 S.D.(n¼ 3); np o0.05 and nn po 0.01 compared with the control cells; p o 0.05 and p o 0.01 compared with the H2O2-treated cells.

4. Discussion As one of the most important peripheral vascular ischemic diseases, TAO is a common in vascular surgery. Due to its unclear pathogenesis, the treatment of TAO has been very difficult until now. Vascular endothelium is the physical barrier that separates the circulating blood from underlying tissue. It is essential for maintaining vascular tone and blood pressure by promoting anticoagulant, antiatherosclerotic, and antithrombotic pathways. It is well known that the initiate pathogenic factor resulting in TAO disease is endothelial cell injury (Zhang, Wei 2005; Xu et al., 2011). Sera from TAO patients proved to be capable of activating HUVECs (Fazeli et al., 2014). Thus to explore the mechanism underlie the endothelial cell injury is helpful to control this kind of disease. Xu et al. firstly incubated isolated HUVECs with Atorvastatin for 4 h, then with cigarette extract for 12 h, they found that Atorvastatin can protect endothelial function from cigarette extract. These results offer new approaches for the control of TAO disease (Xu et al., 2011). The ECV304 cell line has been proved to possess many characteristics of normal endothelial cells, which is often used in cardiovascular research in vitro (Liu et al., 2009). Using the ECV304 cell damaged by H2O2 (final concentration 380 μmol/L) incubation Cui Leisun et al. have ever proved that Jiajianqingying Recipe is the effective agent to TAO (Ming-Chao et al., 2008). Although these advances, it is still lack of an universally accepted in vitro cell model of TAO disease (MingChao et al., 2008; Zhang et al., 2009; Xu et al., 2011; Fazeli et al., 2014). In our preliminary experiments, we compared cell viability of ECV304 after different periods (3, 6, 12, and 24 h) of incubation with 300 mM H2O2 (data not shown in text), only the cells of 3 h incubation with 300 mM H2O2 showed the viability of about 50%. Thus the concentration of H2O2 (300 mM) for 3-h incubation was selected as TAO cellular model.

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Fig. 3. Protective effects of SFI on H2O2-induced apoptosis in ECV304 cells. Flow cytometry (A and B) and AO–EB staining (C) (  400) detected the apoptosis of ECV304 cell in control, H2O2 and 20, 30, 40 μl/ml SFI groups, a representative experiment out of three is shown; SFI inhibited ECV304 cells apoptosis induced by 300 μM H2O2 in a concentration-dependant manner. ECV304 cells pretreated with 20, 30, and 40 μl/ml SFI, respectively, followed by the treatment of 300 μM H2O2.White arrows indicate early apoptotic cells and black arrows show late apoptotic cells. Data are presented as the mean 7 S.D.(n¼ 3). np o 0.05 and nnp o 0.01 compared with the control; p o0.05 and p o 0.01 compared with the H2O2-treated cells [AO–EB staining, Bar ¼ 50 μm (C)].

Shenfu injection is prepared from traditional Chinese medicines red ginseng and aconite root. It is a famous important Chinese traditional remedy used for the treatment of various diseases especially for cardiac diseases (Gu et al., 2009). It can decrease the extent of ischemia-reperfusion injury to the heart, kidney and liver (Yang et al., 2003; Zhu et al., 2006). Recently, SFI is shown to have a significant therapeutic effect on experimental TAO model rats (Hong et al., 2011). In present experiments, although SFI (20, 30, and 40 μl/mL) alone did not affect the viability of ECV304 cells (Fig. 1B), the decreased cell viability resulted from H2O2 was significantly attenuated by SFI pretreatment in a concentration-dependent manner (Fig. 1C). These results suggest that SFI protected ECV304 cells from H2O2 oxidation and may be a promising candidate recipe for TAO treatment. Increased lipid peroxidation may participate in the VEC damage, and markedly reduced antithrombotic function of VEC in TAO patients (Zhang and Wei, 2005). Oxygen free radical and lipid peroxide response which might damage vascular endothelial cell in TAO markedly increased and the detection of these substances might provide complementary evidences for syndrome differentiation of TAO (Ge et al., 1993). Excessive reactive oxygen species can cause toxicity, which are controlled by antioxidant enzymes and small-molecule antioxidants. As one of the main antioxidant enzymes, superoxide dismutase (SOD) protects cells from oxidative stress-induced injury (Iacobazzi et al., 2014). SOD mainly scavenges superoxide anion radical (O2 ), by accelerating its conversion to H2O2, and then glutathione peroxidase (GPx) transforms H2O2 into water. Malondialdehyde (MDA) is a lipid peroxidation marker that is invariably employed to evaluate the oxidative and antioxidant status in cells (Li et al., 2013). In present

experiment (Fig. 2), the incubation of ECV304 cells with 300 μM H2O2 for 3 h decreased significantly the activities of total SOD and GSH-Px and increased the MDA levels in H2O2 treated group. These results conform to some of the characteristic of TAO disease (Ge et al., 1993; Zhang and Wei, 2005). Shenfu Decocton from which SFI is originated can increase SOD activity, decrease MDA level, and improve cardiac function, tissue morphology and ultrastructure changes in acute experimental myocardial ischemia induced by the ligation of coronary artery in rats (Shang et al., 2004; Chen et al., 2005). Our previous data indicates the mech anisms that SFI protects renal structure and function against acute renal ischemia-reperfusion injury may be involved in increasing SOD activity, scavenging directly oxygen free redicals (Yang et al., 2003). Similarly, in this study, SFI significantly enhanced the activities of SOD and GSH-Px, while decreased MDA levels of ECV304 cells (Fig. 2), which suggests that SFI has the ability to protect ECV304 cells from oxidative toxicity induced by H2O2 and may play a role for the clinical treatment of TAO disease. Apoptosis of vascular endothelial cells is an important cause of vascular endothelium damage which is closely associated with the pathogenesis of TAO. H2O2 has been widely used as a model of exogenous oxidative stress in the studies of apoptosis (Chen et al., 2011; Iacobazzi et al., 2014). In this study, the exposure of ECV304 cells to 300 mM H2O2 for 3 h induced ECV304 cells apoptosis (Fig. 3), the percentage of apoptotic cells were significantly increased in both early and late stages of apoptosis. It is comparable to the previous reports (Sun et al., 2012; Ni et al., 2013). As the major components of SFI, ginsenosides compound has obvious protective effects on cardiacmyocytes against apoptosis

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Fig. 4. Effect of SFI on the protein expressions of caspase-3(A), Bcl2 and Bax (B) in ECV304 cells. ECV304 cells pretreated with 20, 30, and 40 μl/ml SFI for 3 h respectively, followed by the treatment of 300 μM H2O2 for 3 h. The cell lysate was subjected to electrophoresis on 12% SDS-PAGE with subsequent enzyme immunoassay using ECL. β-Actin was used as internal control. Representative immunoblots and corresponding cumulative data induced by SFI treated cells and H2O2-treated cells. Each point represents the mean 7 S.D. of three different experiments. np o 0.05 and nnp o 0.01 compared to control and po 0.05 and po 0.01 compared to H2O2-treated cells.

induced by hypoxia/reoxygenation injury, whose mechanisms probably involve the inhibition of down-regulation of Bcl-2 protein levels and sequential activation of caspase-3 (Gu et al., 2009). SFI can also inhibit the proliferation and induce the apoptosis of PC-3 cells, which may due to its upregulation of the p53 mRNA expressions (Lu et al., 2014; Sun et al., 2014). Xiao et al. (2013) observed that SFI treatment could effectively reverse the change of caspase-3/7 activity, and reduce significantly apoptotic cardiac myocyte induced by Fupian injection in vitro. Here, we first demonstrated that a marked dose-dependent decrease in both early and late stages of apoptosis was obvious in ECV304 cells after 20, 30, and 40 μl/mL SFI treatment when compared with H2O2 group. Furthermore, the results from AO/EB double staining showed that ECV304 cells in control group appear to be green nuclei and intact structure when treated with 300 μmol/ L H2O2 for 3 h, the ECV304 cells presented shrinkage, membrane blebbing, chromatin condensation, and formation of apoptotic bodies. However, the ECV304 cells in 20, 30, and 40 μl/mL SFI groups displayed few morphological changes of apoptosis in a concentration-dependent manner. It indicates that SFI significantly inhibited ECV304 cells apoptosis in a concentration-dependent manner. In order to further elucidate the molecular mechanism underlying SFI against H2O2-induced apoptosis, the B cell lymphoma-2 (Bcl-2) proteins family that involved in the mitochondrial apoptosis pathway was examined in this experiment. Moreover, Bax and Bcl-2 acts as a pro-apoptotic anti-apoptotic protein respectively. It is considered that Bax may control the mitochondrial permeability and lead to the activation of caspase-3 and apoptosis. But the influences of Bax can be withstanded by Bcl-2 (Saiprasad et al., 2014). Here, western blot results showed that the treatment of ECV304 cells with 300 μM H2O2 significantly increased the protein expressions of Bax and caspase-3, while significantly decreased Bcl-2 levels. SFI significantly prevented the H2O2-induced decrease of Bcl-2, and the increase of Bax protein expression (Fig. 4), which indicates that the influence of SFI against H2O2-induced apoptosis may be associated with the modulation of Bax and Bcl-2 protein levels. In the meantime, caspase plays a key

role in the execution of apoptosis (Florentin and Arama, 2012). Caspase-3 mediates apoptosis from both extrinsic and intrinsic pathways. This study demonstrates that caspase-3 protein expression increased in ECV304 cells after 3 h of treatment with 300 μM H2O2. When pretreated with SFI, the effect of H2O2 on the expression of caspase-3 in ECV304 cells was markedly inhibited in a concentration-dependent manner. It indicates that the caspase pathway may be involved in the protective effect of SFI against H2O2-induced apoptosis in ECV304 cells. In conclusion, our findings provide the first evidences that SFI has a protective effect on the damaged ECV304 cells by H2O2 treatment, which is likely involved with the regulation of the activities of SOD and GSH-Px, the levels of intracellular MDA, as well as the protein expressions of Bax, Bcl2 and caspase3. It will provide scientific basis for the clinical application of SFI treating TAO disease.

Acknowledgements This work was supported by National Natural Science Foundation of China (No. 81260504), Educational Commission of Jiangxi Province of China (No. GJJ12073) and Health Department of Jiangxi Province of China (No. 20132018).

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