Tanshinone IIA reduces apoptosis induced by hydrogen peroxide in the human endothelium-derived EA.hy926 cells

Journal of Ethnopharmacology 143 (2012) 100–108

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Tanshinone IIA reduces apoptosis induced by hydrogen peroxide in the human endothelium-derived EA.hy926 cells Lian-qun Jia a,b, Guan-lin Yang a,b,n, Lu Ren a, Wen-na Chen a, Jun-yi Feng c, Yang Cao a, Lin Zhang a, Xue-tao Li a, Ping Lei a a

Department of Biochemistry and Molecular Biology, Liaoning University of Traditional Chinese Medicine, Shenyang 110847, China Key Laboratory of Ministry of Education for TCM Viscera-State Theory and Applications, Ministry of Education of China (Province-Ministry Co-construct), Shenyang 110847, China c College of Engineering and Computer Science, California State University, Fullerton, CA 92831, USA b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 13 February 2012 Received in revised form 16 May 2012 Accepted 6 June 2012 Available online 28 June 2012

Ethnopharmacological relevance: Salvia Miltiorrhiza Bunge (also known as herb Danshen in Chinese) is a widely used Chinese herbal medicine. Tanshinone IIA (TSN IIA) is considered to be the most important bioactive ingredient in Danshen and exhibits an anti-atherosclerotic activity. Aim of study: To evaluate the protective effect of TSN IIA on the human endothelial EA.hy926 cells injured by hydrogen peroxide in vitro and its possible mechanism. Materials and methods: The EA.hy926 cells were incubated for 24 h with different concentrations of TSN IIA (5, 10 and 20 mg/mL ) or DMEM. Subsequently, cells were treated with 300 mmol/L H2O2 for another 4 h. Then, the percentage of cell viability was evaluated by 3-(4, 5-di-methylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. The apoptosis of EA.hy926 cells was detected by flow cytometry with AnnexinV-FITC/PI double staining and laser scanning spectral confocal technique. The generation of intracellular reactive oxygen species (ROS) generation was analyzed by flow cytometry. The mRNA expressions of caspase-3, Bcl-2 and Bax were tested by real time-reverse transcription polymerase chain reaction (real time RT-PCR). The protein expression of Bcl-2 and Bax was determined by Western blotting. MDA levels, NO production, LDH leakage, and SOD as well as caspase-3 activities were also measured using standard methods. Results: Loss of cell viability and excessive cell apoptosis were observed in EA.hy926 cells after 4 h of challenge with H2O2 (300 mmol/L). However, cell apoptosis was attenuated in different concentrations of TSN IIA (5, 10 and 20 mg/mL) pretreated cells. Furthermore, TSN IIA markedly inhibited the elevation of ROS evoked by H2O2. Real time RT-PCR and Western blotting analysis showed that TSN IIA significantly decreased the expressions of pro-apoptotic proteins (Bax and caspase-3) while significantly increased the expression of anti-apoptotic protein Bcl-2, and resulted in obvious reduction of Bax/Bcl-2 ratio in EA.hy926 cells induced by H2O2. Conclusion: These observations provide preliminary evidence that TSN IIA protects EA.hy926 cells against H2O2 damage, which is mainly associated with the ROS generation, followed by the imbalance of the Bax/Bcl-2 ratio, and caspase-3 activation leading to apoptosis. & 2012 Elsevier Ireland Ltd. All rights reserved.

Keywords: Tanshinone IIA Apoptosis Reactive oxygen species Bax/Bcl-2 ratio Caspase-3

1. Introduction

Abbreviations: TSN IIA, Tashinone IIA; MTT, 3-(4, 5-di-methylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide; ROS, reactive oxygen species; Real time RT-PCR, real time-reverse transcription polymerase chain reaction; NO, nitric oxide; H2O2, hydrogen peroxide; FBS, fetal bovine serum; DMSO, dimethyl sulfoxide; SOD, super-oxide dismutase; LDH, lactate dehydrogenase; MDA, malonaldialdehyde; PI, propidium iodide n Corresponding author at: Department of Biochemistry and Molecular Biology, Liaoning University of Traditional Chinese Medicine, Shenyang 110847, China. Tel.: þ86 24 3120 7028; fax: þ86 24 3120 7014. E-mail address: [email protected] (G.-l. Yang). 0378-8741/$ - see front matter & 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2012.06.007

Endothelial injury is considered to be an initial step in the pathogenesis of atherosclerosis (Higashi et al., 2009), which is also the pathological basis of various cardiovascular and cerebrovascular disorders (Katz et al., 2001). Growing evidence reveals a relationship between oxidative stress and endothelial function and oxidative stress has been recognized as a key mechanism in the development of vascular damage, particularly atherosclerosis (Minuz et al., 2006). There are several possible mechanisms for the oxidative stress impairment of endothelial function in cardiovascular diseases, including enhanced production of reactive oxygen species (ROS) and decreased release of

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nitric oxide (NO), as well as an attenuated antioxidant system (Cai and Harrison, 2000; Deanfield et al., 2007). Oxidative stress may result in apoptosis of endothelial cell, which contributes to atherogenesis and other vascular diseases (Harrison et al., 2003; Quagliaro et al., 2003; Sudoh et al., 2001). A number of investigators have reported that apoptosis of endothelial cell can be triggered by ROS (Irani, 2000; Li and Shah, 2004). Furthermore, apoptotic cell death following injury of vascular endothelium is assumed to play an important role in the pathogenesis of atherosclerosis (Cines et al., 1998; Falk, 2006). Tanshinone IIA (TSN IIA) is the most abundant diterpene quinone in Salvia Miltiorrhiza Bunge (Danshen). It is a widely prescribed traditional herbal medicine used for the prevention and treatment of atherosclerosic disease (Wang et al., 2003, 2010). It is also considered to be the most important bioactive ingredient in Danshen and exhibits a variety of cardiovascular and cerebrovascular activities. TSN IIA provides beneficial effects toward atherosclerosic disease through several pathways. Accumulating studies demonstrated that TSN IIA possesses many biological and pharmacologic properties primarily depending on its anti-oxidative effects (Lin et al., 2006; Tang et al., 2007). Although TSN IIA has been proved to have anti-oxidant effects on preventing endothelial cell from oxidative stress-triggered damage and apoptosis (Lin et al., 2006; Wu et al., 2007), many of its anti-oxidant and anti-apoptotic mechanisms remain to be demonstrated. And little data is available about its anti-atherosclerotic role and mechanisms in human endothelium-derived EA.hy926 cells. In the present study, we examined the protective effects of TSN IIA on hydrogen peroxide (H2O2)-induced apoptosis of EA.hy926 cells and investigated the possible mechanisms of action involved.

2. Materials and methods 2.1. Drug and reagents TSN IIA was purchased from the Chinese Institute for Drug and Biological Product Control (Beijing, China) and then was dissolved in DMSO (final concentration 0.2 mL/L). The solution was filtered through a 0.22 mm micropore filter and stored at 4 1C. DMEM medium, trypsin, and fetal bovine serum (FBS) were purchased from Hyclone (Hyclone Logan, UT). 3-(4, 5-dimethyl-thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT), dimethyl sulphoxide (DMSO), penicillin, streptomycin were obtained from Sigma (St. Louis, MO, USA). Rabbit anti-human Bcl-2 antibody, rabbit antihuman Bax antibody and goat anti-rabbit IgG were supplied by Santa Cruz Biotechnology (Santa Cruz, CA, USA). Super-oxide dismutase (SOD), NO, lactate dehydrogenase (LDH), malonaldialdehyde (MDA) assay kits, H2O2, Annexin V fluorescein isothiocyanate (FITC) and propidium iodide (PI) apoptosis detection kits were produced by Nanjing Key-Gen Biotech Co., Ltd. (Nanjing, China). The SYBR ExscriptTM RT-PCR Kit and Trizol reagent were purchased from TaKaRa Bio Inc. (Dalian, China). RIPA Lysis Buffer, caspase-3 activity kit, ROS assay kit was produced by Beyotime Institute of Biotechnology (Nantong, China). All other reagents commercially available were of the highest purity. 2.2. Cell culture EA.hy926 human vascular endothelial cell line was purchased from American Type Culture Collection and maintained in highglucose DMEM medium containing 10% fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin at 37 1C, 5% CO2. Cells in logarithmic growth phase were used for further experiments. Before experimental intervention, confluent-cultured

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cells were preincubated for 24 h in serum-starved medium including DMEM with 1% fetal bovine serum. 2.3. Oxidative damage induced by H2O2 EA.hy926 cells were cultured in 24-well plates at a density of 2  105 cells/ml for 24 h. TSN IIA (5, 10 and 20 mg/mL) was dissolved in DMSO (no more than 0.1% in v/v) and was added into the wells for 24 h incubation, and then the cells were exposed to 300 mmol/L H2O2 for another 4 h except the normal control. 2.4. Measurement of cell viability The viability of EA.hy926 cells was measured by a colorimetric assay using MTT. The assay was performed by seeding EA.hy926 cells in the concentration of 1  104 cells/well in 96-well plate. After different concentrations of TSN IIA (5, 10 and 20 mg/mL) in DMEM medium were added into the wells to incubate 24 h, cells were exposed to 300 mmol/L H2O2 for further 4 h. Then MTT solution (5 mg/mL) was added to each well, and the plate was incubated 4 h at 37 1C. After incubation, medium was removed and 150 mL DMSO was added to each well for formazan solubilization. Finally, the optical density (OD) of each well was measured on a microplate reader at 570 nm. The OD of formazan formed in untreated cells was taken as 100% viability. 2.5. Measurement of intracellular ROS Intracellular ROS formation was quantified using redox-sensitive dye 2,7-dichlorodihydrofluorescin diacetate (DCFH-DA) and flow cytometric analysis. Briefly, after exposure to 300 mmol/L H2O2 for 4 h, cells were harvested with trypsin and washed with phosphate buffered saline (PBS) twice. Then, 1  106 cells were incubated with 10 mmol/L DCFH-DA for 20 min at 37 1C. The fluorescence of 2,7-dichlorofluorescein (DCF) was detected using flow cytometry and all the measurements were repeated in triplicate. 2.6. Determination of NO production and LDH leakage The production of NO was tested by measuring the accumulation of nitrites in the supernatant of cells. In brief, 100 ml Griess reagent was added to 100 ml of sample. After incubating at room temperature for 10 min, the OD value was measured at 550 nm with a Microplate Reader. To evaluate the effects of TSN IIA on LDH leakage, EA.hy926 cells were treated in preparation for a cell viability assay. At the end of incubation, the supernatant was collected and LDH leakage was measured using the assay kit according to the manufacturer’s instructions. 2.7. Evaluation of MDA levels and SOD activities After exposured to 300 mmol/L H2O2 for 4 h, EA.hy926 cells were collected to measure MDA levels and SOD activities. Briefly, cells were washed twice with PBS and lysed with lysis buffer. Then the homogenate was centrifuged at 12,000g at 4 1C for 15 min. The MDA level and SOD activities in the supernatant were measured by spectrophotometric methods. 2.8. Determination of apoptotic cells Double staining for Annexin V-FITC and propidium iodide (PI) was performed to estimate the apoptotic rate of EA.hy926 cells. Briefly, after incubated with various concentrations of TSN IIA for 24 h, EA.hy926 cells were treated with 300 mmol/L H2O2 for another 4 h.

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Subsequently, the cells were trypsinized and washed twice with PBS, centrifuged at 800 rpm for 5 min. Then, 1  106 cells were suspended in binding buffer and double-stained with Annexin V-FITC and PI for 30 min at room temperature. After that, the fluorescence of each sample was quantitatively analyzed by FACS calibur flow cytometer and CellQuest software. The results were interpreted as follows: cells negative for both PI and Annexin V-FITC staining were considered normal live cells. PI-negative, Annexin V-FITC-positive stained cells were considered in early apoptosis. PI positive, AnnexinV-FITC-positive stained cells were considered in late apoptosis. Then, double stained samples were further examined with a Leica TCS SP2 laser scanning spectral confocal system (Leica Microsystems GmbH, Heidelberg, Germany). An Argon laser was used to excite Annexin V-FITC (488 nm) and PI (586 nm) fluorochromes. 2.9. Total RNA isolation and real time RT-PCR Total RNAs were extracted from EA.hy926 cells of each group by using Trizol reagent (TaKaRa, Dalian, China). The purity and the concentration of the total RNAs were measured by ultraviolet spectrophotometry. SYBR Green I PCR was performed in duplicate for each sample using the ABI 7500 PCR Sequence Detector (Biometra) and the SYBR ExscriptTM RT-PCR Kit (TaKaRa, Dalian, China). The following primers were used: bcl-2: (forward primer, 50 -CACCTGTGGTCCACCTGAC-30 and reverse primer, 50 -AGGGCCAAACTGAGCAGAG-30 ), fragment length was 406 bp; Bax: forward primer, 50 -TGCTTCAGGGTTTCATCCA-30 and reverse primer, 50 -GACACTCGCTCAGCTTCTTG-30 , fragment length was 111 bp; caspase-3: forward primer, 50 -GTGGCATTGAGACAGAC-30 and reverse primer 50 -GGCACAAAGCGACTG-30 , fragment length was 155 bp. b-actin was amplified (forward primer, 50 -AGTTGCGTTACACCCTTTC-30 and reverse primer, 50 -TGTCACCTTCACCGTTCC-30 , fragment length was 152 bp) and used as a standard for each PCR analysis. These primers were designed and synthesized by TaKaRa Biotechnology (Dalian, China). The conditions for the PCR were as follows: 50 cycles of denaturation at 94 1C for 30 s, annealing at 60 1C for 30 s, and extension at 72 1C for 30 s. The fold-change in gene expression was determined by the DDCT method. 2.10. Western blotting EA.hy926 cells were collected and total proteins were extracted by RIPA Lysis Buffer (Beyotime Institute of Biotechnology, Shanghai). Protein concentration was measured by a quantification kit (Boster Biological Technology, Wuhan). Protein samples were resolved in 12% polyacrylamide gels, then transferred to PVDF membrane and blocked with 5% nonfat dairy milk in Tris-buffered saline with 0.05% Tween-20. Membranes were incubated with the following primary antibodies: rabbit anti-human Bcl-2(dilution 1:500), Bax antibody (dilution 1:500) (Santa Cruz, CA, USA) followed by HRP-conjugated secondary antibody (dilution 1:1000) (Santa Cruz, CA, USA) and developed with ECL reagent (Beyotime Institute of Biotechnology, Shanghai).

caspase-3 was assayed at an absorbance of 405 nm. Caspase-3 activity was expressed as a percentage of the control. 2.12. Statistical analysis All data are presented as the mean7standard deviation (S.D.). One-way ANOVA followed by Student’s t test was used for the statistical analysis by employing SPSS 13.0 software. Difference at the Po0.05 level was considered statistically significant.

3. Results 3.1. Effect of TSN IIA on the viability of EA.hy926 cells injured by H2O2 The effect of TSN IIA on the viability of EA.hy926 cells injured by H2O2 was measured by MTT assay. As showed in Fig. 1, after exposure to 300 mmol/L H2O2 for 4 h, the viability of EA.hy926 cells decreased to 49.89% of the control group, while on pretreatment with TSN IIA (5, 10 and 20 mg/mL), the cell viability markedly increased to 60.43%, 72.48% and 85.12% of the control group in a dose-dependent manner. These findings suggest that TSN IIA protects EA.hy926 cells from oxidative stress-related cellular injuries. 3.2. Effect of TSN IIA on NO release, LDH leakage, MDA production, and activities of total SOD in EA.hy926 cells As shown in Fig. 2, incubation of the cells with 300 mmol/L H2O2 for 4 h caused a significant decrease in SOD activity and NO release while significant increase in LDH leakage and MDA equivalents compared with control group (all Po0.01). In contrast, preincubation of the cells with TSN IIA (5, 10 and 20 mg/mL) considerably attenuated the decreased SOD activity and NO level while prominently inhibited the increased LDH leakage and MDA equivalents induced by H2O2 in a dose-dependent manner. The result suggests that TSN IIA increases NO release, represses LDH leakage, MDA production, and restores the activities of total SOD in EA.hy926 cells. 3.3. Effect of TSN IIA on the generation of oxidant-induced ROS in EA.hy926 cells To determine the effect of TSN IIA on H2O2-generated ROS in EA.hy926 cells, the intracellular ROS level was determined by DCFH-DA probe oxidation. As shown in Fig. 3, exposure to 300 mmol/L H2O2 alone increased ROS levels of EA.hy926 cells

2.11. Analysis of caspase-3 activation To further examine the effects of TSN IIA on H2O2-induced apoptosis, caspase-3 activity was determined by a colorimetric assay according to the manufacturer’s protocol. Briefly, EA.hy926 cells were rinsed with cold PBS and then lysed with lysis buffer. The lysate was centrifuged at 16,000g at 4 1C for 15 min. After mixing 10 ml of protein from the cell lysate, 80 ml of reaction buffer, and 10 ml of caspase-3 substrate together in 96-well microtiter plates were incubated at 37 1C for 4 h. The activity of

Fig. 1. Effect of TSN IIA on EA.hy926 cells viability injured by H2O2. Cell viability was assayed by the MTT method (n ¼6). All data are presented as means7 SD. nn P o0.01 vs. control group; #Po 0.05 vs. H2O2 group; ##P o0.01 vs. H2O2 group.

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Fig. 2. Effects of TSN IIA on nitric oxide (NO) release (A), malondialdehyde (MDA) equivalents (B), lactate dehydrogenase (LDH) leakage (C) and the activities of total superoxide dismutase (SOD) (D) in EA.hy926 cells under oxidative stress induced by H2O2. All data are presented as means 7SD, n ¼6. nnP o0.01 vs. control group; # P o 0.05 vs. H2O2 group; ##Po 0.01 vs. H2O2 group.

about 2.5 fold. The increase was partially prevented by preincubation with TSN IIA in a dose-dependent manner. These observations suggest that TSN IIA could scavenge the generation of oxidant-induced ROS in EA.hy926 cells. 3.4. Effect of TSN IIA on H2O2-induced apoptosis in EA.hy926 cells Laser scanning microscopy technique and flow cytometry were used to evaluate the effect of TSN IIA on oxidant-induced apoptosis in EA.hy926 cells. As shown in Fig. 4, exposure to 300 mmol/L of H2O2 induced significant elevations of apoptosis (Po0.01 vs. control group), the cell apoptosis rate was increased about 4 fold compared to the control group (24.2671.51% vs. 5.59 70.62%). However, TSN IIA significantly attenuated the EA.hy926 cells injury by H2O2 stimuli. TSN IIA reduced apoptotic cells in a dose-dependent manner, and after pretreatment with TSN IIA 5, 10 and 20 mg/mL, the apoptotic rate of EA.hy926 cells induced by H2O2 decreased to 16.5771.11% (Po0.01 vs. H2O2 group), 10.3070.72% (Po0.01 vs. H2O2 group) and 6.2570.23% (Po0.01 vs. H2O2 group), respectively. These data suggest that TSN IIA protects EA.hy926 by attenuating apoptosis induced by H2O2. 3.5. Effect of TSN IIA on the mRNA expression levels of caspase-3, Bcl-2 and Bax To determine whether the inhibitory effects of TSN IIA on the apoptosis in EA.hy926 cells were related to the modulation of

gene expression, the transcription levels of caspase-3, Bcl-2 and Bax mRNA were investigated by using real-time RT-PCR. Fig. 5 showed that 300 mmol/L H2O2 significantly increased mRNA expression of caspase-3 and Bax, while significantly decreased mRNA expression of Bcl-2 compared to the control group. However, EA.hy926 cells preincubated with TSN IIA at concentrations of 5, 10 and 20 mg/mL had markedly enhanced Bcl-2 mRNA expression while markedly inhibited caspase-3 and Bax mRNA expression in a dose-dependent manner. 3.6. Effect of TSN IIA on protein expression levels of Bcl-2 and Bax as well as caspase-3 activation in EA.hy926 cells The expression of the proapoptotic factor Bax and that of the antiapoptotic factor Bcl-2 were further analyzed by Western blotting method. Fig. 6(A and B) showed that incubation with H2O2 resulted in Bax expression increased and Bcl-2 expression reduced significantly compared to the control group. However, the presence of 5, 10 and 20 mg/mL TSN IIA demonstrated antiapoptotic activity via increasing Bcl-2 expression and inhibiting Bax expression. Furthermore, as indicated in Fig. 6C, incubation with H2O2 resulted in obvious increase of Bax/Bcl-2 ratio, while TSN IIA decreased Bax/Bcl-2 ratio remarkably in a dose-dependent manner. The effects of TSN IIA on caspase-3 activities were determined by a colorimetric assay. As presented in Fig. 6D, the exposure to 300 mmol/L H2O2 alone increased caspase-3 activity to 222.7710.6% relative to the control group (po0.01), while

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Fig. 3. Effects of TSN IIA on ROS generation in EA.hy926 cells under oxidative stress induced by H2O2. Cells were continued incubated with the cell permeable DCFH-DA (10 mmol/L) for 20 min and analyzed immediately by a flow cytometer. (A–E) represented histogram of fluorescence measured by flow cytometry in different groups. (F) The results of mean fluorescence intensity measured by DCFH analysis are demonstrated. All data are presented as means 7 SD, n¼ 3. nnPo 0.01 vs. control group; #P o 0.05 vs. H2O2 group; ##P o 0.01 vs. H2O2 group.

preincubation with TSN IIA at concentrations of 5, 10 and 20 mg/ mL markedly attenuated the increase in caspase-3 activity in a dose-dependent manner.

4. Discussion Salvia Miltiorrhiza Bunge (also known as herb Danshen in Chinese) is a widely used Chinese herbal medicine. The chemical constituents from roots of Danshen are classified into two major categories: water-soluble compounds and lipophilic diterpenoid quinines (Han et al., 2008). The major diterpenes in Danshen include cryptotanshinone, tanshinone I, tanshinone IIA and dihydrotanshinone (Wang et al., 2003). Among them, TSN IIA had been shown to possess various pharmacological activities and considered to be the most important bioactive ingredient in Danshen (Li et al., 2008). Endothelial injury is considered to be an initial event in the development of atherosclerosis. Some major risk factors for atherosclerosis such as oxidized low density lipoprotein, angiotensin II as well as ROS, could promote endothelial cell apoptosis and thereby may contribute to the initiation of

atherosclerosis (Choy et al., 2001). Oxidative stress has been implicated in pathological processes associated with atherosclerosis and other cardiovascular diseases. Hence, inhibition of endothelial cell apoptosis induced by oxidative stress is an essential therapeutic strategy for atherosclerosis. Although TSN IIA has been proved to elicit a series of biologic effects through its anti-oxidative property, the mechanism underlying these effects is still unclear. Our previous investigation revealed that TSN IIA had a protective effect on endothelial cells by intervening the TLR4/NF-kB inflammatory signal pathway. (Jia et al., 2011a, 2011b). In the present study, we found that TSN IIA significantly attenuated oxidative stress and inhibited apoptosis in EA.hy926 cells, which might contribute to its anti-atherosclerotic action. Normal endothelial cells maintain a delicate balance in the vasculature between vasodilation and vasoconstriction, antiinflammation and pro-inflammation, and also antioxidation and pro-oxidation (Higashi et al., 2009; Vanhoutte, 1997). Vascular endothelial cells are in constant contact with steady-state levels of oxidative metabolites, which are increased in a number of pathophysiological processes that affect the blood vessel such as atherosclerosis (Hermann et al., 1997). In this study, the human

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Fig. 4. Effect of TSN IIA on oxidant-induced apoptosis in EA.hy926 cells. The apoptosis of EA.hy926 cells was measured by the confocal laser scanning microscopy and flow cytometry. (A) showed representative thin section confocal fluorescence micrographs of EA.hy926 cells. B showed the proportion (%) of cell number in each quadrant. Lower left quadrant (absence of both markers) indicated viable cells; upper left quadrant (PI positive) indicated cellular necrosis. The total apoptotic cells (early and latestage apoptosis) were represented by the right side of the panel (Annexin V staining alone or together with PI). Data are presented as means 7 SD, n ¼3. nnPo 0.01 vs. control group; #Po 0.05 vs. H2O2 group; ##P o0.01 vs. H2O2 group.

Fig. 5. Effect of TSN IIA on the mRNA expression levels of caspase-3, Bcl-2 and Bax in EA.hy926 cells. The mRNA expression levels of caspase-3, Bcl-2 and Bax were estimated by quantitative real-time reverse-transcription PCR. Results were shown as relative expression ratio of caspase-3, Bcl-2 and Bax mRNA in EA.hy926 cells with respect to the control group (equals to 1 by definition) and normalized by b-actin reference gene expression. Bars represent the means7 SD, n¼3. nnP o0.01 vs. control group; #Po 0.05 vs. H2O2 group; ##P o0.01 vs. H2O2 group.

endothelial EA.hy926 cells line was used as a model to discuss the anti-oxidative and anti-apoptotic effect of TSN IIA in vitro. The EA.hy926 cells line was generated in 1983 by the fusion of human umbilical vein endothelial cells (HUVECs) with the human lung carcinoma cell line A549 (Bouı¨s et al., 2001). These hybrid cells have been shown to possess many characteristics of normal endothelial cells, so they are commonly used in cardiovascular studies (Dichtl et al., 2003; Piqueras et al., 2007; Rival et al., 2002). The endothelial generation of ROS is important both physiologically and in pathogenesis of many cardiovascular disorders (Li and Shah, 2004). There is substantive evidence that ROS signal stimulate endothelial cell activities and induce cell injury and

apoptosis by oxidant modification of proteins and carbohydrates, lipid peroxidation, and DNA strand nicks (Lum and Roebuck, 2001). Our results showed that formation of intracellular ROS in EA.hy926 cells under H2O2-induced oxidative stress was significantly inhibited by TSN IIA in a dose-dependent manner. Moreover, TSN IIA noticeably decreased the apoptotic rate of H2O2-induced EA.hy926 cells evidenced by Annexin V-FITC/PI double staining. These results suggested that the protective effect of TSN IIA may be associated with the inhibition of intracellular ROS production. The endothelium plays a pivotal role in control of vascular tone by releasing several vasoactive substances, such as NO. Endothelium-dependent relaxation is impaired in animals with

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Fig. 6. Effect of TSN IIA on protein expression levels of Bcl-2 and Bax as well as caspase-3 activation in EA.hy926 cells. (A) Expression levels of Bcl-2. (B) Expression levels of Bax. (C) Ratio of Bcl-2/Bax analysis. (D) The activity of caspase-3. Data are presented as means 7SD, n¼3. **P o 0.01 vs. control group; #Po 0.05 vs. H2O2 group; ##Po 0.01 vs. H2O2 group.

atherosclerosis, which has been linked to a decreased production and/or biological activity of endothelium-derived NO (Greene et al., 1993; Naruse et al., 1994). Oxidative inactivation of NO is regarded as an important cause of its decreased biological activity ¨ (Boger et al., 1996). A balance of endothelium-derived vasodilators, especially NO and ROS could modulates endothelial function. Decreased NO bioavailability induces endothelial dysfunction. Therefore an imbalance of NO and ROS, so-called oxidative stress, is involved in endothelial dysfunction through the inactivation of NO (Higashi et al., 2009). Our results showed that TSN IIA increased the release of NO in EA.hy926 cells under H2O2-induced oxidative stress in dose-dependent manner. The susceptibility of vascular cells to oxidative stress is a function of the overall balance between the degree of oxidative stress and the antioxidant defense capability (Yukihito and Yoshizumi, 2004). SOD is a major antioxidant enzyme that protects cells against the oxidative stress injury in the endothelial cells (Lefer and Granger, 2000). The main function of SOD is to remove O2  by greatly

accelerating its conversion to H2O2, then H2O2 is eliminated by glutathione peroxidase (GPx) and catalase to water. MDA, as a lipid peroxidation marker, can result in cell membrane breakdown and further cause cell swelling (Chen et al., 2011; Giray et al., 2003). LDH is a sensitive index of the loss of cell membrane integrity. In the present study, TSN IIA increased the concentration of SOD, decreased the level of intracellular MDA and the leakage of LDH activity in the extracellular medium. Apoptosis, also known as programmed cell death (PCD), is generally characterized by distinct morphological characteristics and energy-dependent biochemical mechanisms (Elmore, 2007). The vascular endothelial monolayer serves as a barrier between the bloodstream and the vascular wall. Endothelial cells apoptosis may play a role in atherosclerosis, angiogenesis, vascular remodeling and other pathophysiological states (Graham and Chen, 2001). Some investigations demonstrate that oxidative stress induces cell apoptosis, hypertrophy, proliferation and inflammation through activation of various signaling cascades and redox-sensitive

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transcriptional factors (Higashi et al., 2009). We detected the influence of TSN IIA on EA.hy926 cells apoptosis induced by H2O2. Results of AnnexinV-FITC/PI double staining and laser scanning spectral confocal detection demonstrated that H2O2 induced a significant apoptosis compared to the control group and TSN IIA inhibited EA.hy926 cells apoptosis in a concentration dependent manner, indicated that TSN IIA has an ability to protect EA.hy926 cells from H2O2 induced injury through anti-oxidative approach. In order to probe the mechanism of TSN IIA protecting EA.hy926 cells against H2O2-induced apoptosis, we further examined expressions of apoptosis related proteins, including Bcl-2, Bax and caspase-3. Members of the Bcl-2 family of proteins are critical regulators of the apoptotic pathway and the Bcl-2 family includes both anti-apoptotic molecules such as Bcl-2, Bcl-XL, Bcl-w, Bfl-1, Bag-1, and pro-apoptotic molecules such as Bax, Blk, Bak, Bid and Bad (Zhong et al., 2011). The relative ratio of anti- vs. pro-apoptotic Bcl-2 family proteins has been shown to dictate the ultimate sensitivity or resistance of cells to various apoptotic stimuli (Adams and Cory, 2001; Wang, 1993). The balance of pro-apoptotic Bax and anti-apoptotic Bcl-2 is known to be important in determining cell death or survival (Wang et al., 2010). Bax translocates from the cytosol to the outer mitochondrial membrane, where it can form Bax:Bcl-2 heterodimers and tetramers, leading to pore formation and cytochrome C release and resulting in caspase activation (Su et al., 2005; Thomenius et al., 2003; Wongtongtaira et al., 2011). Some research have shown that the Bax/Bcl-2 ratio plays an key role in determining whether cells undergo apoptosis (Gupta et al., 2002; Schelman et al., 2004). The Bax/Bcl-2 ratio regulates the release of cytochrome C from mitochondria to cytosol, accompanied by the activation of caspase cascade (Kang and Reynolds, 2009). The caspases, critical mediators of apoptosis, are a family of intracellular cysteine proteases with specificity for aspartic acid residues (Li and Yuan, 2008). Caspases-3 is one of the most important executioners, which is capable of cleaving many important cellular substrates, and caspase-3 mediating cell death plays an important role in apoptosis process (Shi, 2002). In our present study, real time RT-PCR and Western blotting showed that TSN IIA was able to stimulate the protein expression of Bcl-2 and constrain the protein expression of Bax, caspase-3, and significantly decreased the Bax/Bcl-2 ratio.

5. Conclusions Our findings underscore the protective effect of TSN IIA on cells apoptosis induced by H2O2 in the EA.hy926 cells. Our results showed that TSN IIA acted as an antioxidant preventing ROS formation, LDH leakage and MDA production, while promoting NO release as well as SOD activities. In addition, our data demonstrated that TSN IIA significantly inhibited EA.hy926 cells apoptosis and revealed the potential mechanism for its anti-apoptosis effect, which may be related to the expression change of Bcl-2, Bax, caspase-3, especially the resume of balance for Bax/Bcl-2 ratio. Our preliminary data suggested that TSN IIA had the anti-oxidative and anti-apoptotic effect in endothelial cells and further studies are underway to investigate its detailed protective mechanism in vivo.

Acknowledgments This work was financially supported by China Postdoctoral Science Special Foundation (201104611), China Postdoctoral Science Foundation (20090451279), Liaoning Province Colleges and Universities Excellent Talents Support Program (LJQ2011100), and Start-up Fund for PhD of Liaoning Province, China (20091052).

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