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Cardiovascular Research Advance Access first published online on September 24, 2008
This version [Corrected Proof] published online on October 22, 2008
Cardiovascular Research, doi:10.1093/cvr/cvn262
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2008. For permissions please email: journals.permissions@oxfordjournals.org

Salvianolic acid B protects human endothelial cells from oxidative stress damage: a possible protective role of glucose-regulated protein 78 induction

Hong-Li Wu1, Yu-Hua Li1, Yan-Hua Lin1, Rui Wang1, Ying-Bo Li1, Lu Tie1, Qian-Liu Song2, De-An Guo3, He-Ming Yu2 and Xue-Jun Li1,*

1 Department of Pharmacology, School of Basic Medical Sciences, Health Science Center, Peking University, Xueyuan Road, Haidian District, Beijing 100083, PR China
2 National Research Institute for Family Planning, Beijing 100081, PR China
3 State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100083, PR China

* Corresponding author. Tel: +86 108 280 2863; fax: +86 108 280 2863. E-mail address: xjli{at}bjmu.edu.cn

Received 16 October 2007; revised 16 September 2008; accepted 21 September 2008

Time for primary review: 44 days


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
 References
 
Aims: The purposes of the present study were to both evaluate the protective effects of Salvianolic acid B (Sal B) and to determine the possible molecular mechanisms by which Sal B protects endothelial cells from damage caused by oxidative stress.

Methods and results: Pretreatment with Sal B markedly attenuated H2O2-induced viability loss, lactate dehydrogenase leakage and apoptosis in human umbilical vein endothelial cells (HUVECs). The mechanism of Sal B protection was studied using two-dimensional gel electrophoresis coupled with hybrid quadrupole time-of-flight mass spectrometry. Database searching implicated that glucose-regulated protein 78 (GRP78), a central regulator for endoplasmic reticulum (ER) stress, was up-regulated in Sal B-exposed HUVECs. GRP78 expression regulation was confirmed by western blot and RT–PCR (reverse transcription–polymerase chain reaction) analyses. Additionally, GRP94, which shares significant sequence homology with GRP78, was also up-regulated in Sal B-treated cells. Sal B caused pancreatic ER kinase (PKR)-like ER kinase (PERK) activation followed by the phosphorylation of eukaryotic translation initiation factor 2{alpha} (eIF2{alpha}) and the expression of activating transcription factor 4 (ATF4). Knockdown of endogenous ATF4 expression partially repressed Sal B-induced GRP78 induction. Further investigation showed that ATF6 was also activated by Sal B. Knockdown of GRP78 by siRNA significantly reduced the protective effects of Sal B.

Conclusion: The results suggest that Sal B induces the expression of GRP78 by activating ATF6 and the PERK–eIF2{alpha}–ATF4 pathway. Furthermore, up-regulation of GRP78 by Sal B may play an important role in protecting human endothelial cells from oxidative stress-induced cellular damage.

KEYWORDS Salvianolic acid B; Oxidative stress; Endothelial cell; GRP78; Endoplasmic reticulum stress


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Funding
 References
 
Endothelial cell death or injury may contribute to the initial endothelial pathophysiological processes, such as angiogenesis, atherosclerosis, and thrombosis.1 Previous studies have indicated that the vascular endothelium was sensitive to reactive oxygen species (ROS) that can cause cell damage and death. Oxidative stress was reported to be a major factor in damaging endothelial cells and has been implicated in endothelial dysfunction occurring via different mechanisms.2 A number of antioxidant compounds have been tested as agents to restore endothelial function. Several prospective antioxidant agents were found to have therapeutic effects on cardiovascular diseases and cancers.3,4

The dried root of Salvia miltiorrhiza is called Danshen. It has been widely used in China and, to a lesser extent, Japan, the United States, and other European countries for the treatment of cardiovascular disorders and cerebrovascular diseases. This herb is an important source of a large number of active natural compounds. Salvianolic acid B (Sal B), a pure water-soluble compound extracted from Danshen, has been studied extensively for its broad pharmacological activities. Previous studies have shown that Sal B protected the brain and heart from ischemia-reperfusion injury by inhibiting the lipid peroxidation and superoxide anion production.5–7 Experimental data have demonstrated that Sal B might inhibit platelet aggregation,8 prevent oxidation of low-density lipoprotein,9 and inhibit tumour necrosis factor-{alpha}-induced matrix metalloproteinase-2 up-regulation.10 Our previous data also indicated that Sal B could prevent cytotoxicity induced by β-amyloid protein.11 Although pharmacological studies indicate that Sal B imparts cardiovascular benefits, the mechanisms underlying its protective effects have not yet been fully understood. In previous studies, a majority of research attributed the pharmacological actions of Sal B mainly to its antioxidant activity. We hypothesized that some other mechanisms could also contribute to its protective effects. Here we studied the mechanisms by which Sal B protects human endothelial cells using a comprehensive proteomic analysis. The results suggest that Sal B protects human endothelial cells from oxidative stress-induced cellular damage by up-regulating glucose-regulated protein 78 (GRP78).


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Funding
 References
 
2.1 Materials
M199 medium, foetal bovine serum (FBS), collagenase I, 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2-tetrazolium bromide (MTT), penicillin and streptomycin were purchased from GIBCOL (Grand Island, NY, USA). The reagent kit used for measurement of lactate dehydrogenase (LDH) was purchased from the Nanjing Institute of Jiancheng Biological Engineering (Nanjing, Jiangsu, China). The Multiparameter Apoptosis 1 HitKit for high content screening (HCS) was from Cellomics, Inc. (Pittsburgh, PA, USA). Hydrogen peroxide and all other chemicals were from Sigma Chemical Co. (St Louis, MO, USA), unless otherwise stated. Antibodies against GRP78, GRP94, PERK [pancreatic ER kinase (PKR)-like ER kinase], phosphorylated PERK, and β-actin were from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and antibodies against phosphorylated eIF2{alpha} (eukaryotic translation initiation factor 2{alpha}), anti-eIF2{alpha} and anti-ATF4 (activating transcription factor 4), were from Signalway Antibody Co., Ltd (Pearland, TX, USA). The ATF6 antibody was purchased from Tianjin Saier Biotechnology (Tianjin, China).

2.2 Extraction and isolation of Salvianolic acid B
Sal B was isolated and identified using previously published methods.11 Its purity was >98% as determined by HPLC analysis. The chemical structure and the chromatogram of Sal B are shown in Figure 1A and B. Sal B was structurally identified based on the spectral data (UV, IR, NMR, MS, and CD).12


Figure 1
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  		Figure 1 The protective effects of Salvianolic acid B (Sal B) on H2O2-induced cytotoxicity in human umbilical vein endothelial cells (HUVECs). (A) The chemical structure of Sal B. (B) The chromatogram of Sal B. (C) Effects of Sal B on the viability of HUVECs treated with H2O2. HUVECs were exposed to various concentrations of Sal B (10–6–10–8 M) for 24 h. After pretreatment, cells were treated with H2O2 (500 µM) for 4 h and cell viability was determined by MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-tetrazolium bromide] assay. (D) Protective effects of Sal B on lactate dehydrogenase (LDH) leakage induced by H2O2 in HUVECs. Cells were pretreated with various concentrations of Sal B (10–8–10–6 M) for 24 h prior to incubation with 500 µM H2O2 for 4 h, which was followed by measuring of LDH leakage as described in Methods. Values are expressed as the mean ± SD of three independent experiments, n = 6. ##P < 0.01, vs. H2O2-free group (Control); **P < 0.01, vs. H2O2 alone group.

 
2.3 Cell culture and treatment
The investigation conformed to the principles outlined in the Declaration of Helsinki.13 Human umbilical vein endothelial cells (HUVECs) were isolated from umbilical cords and cultured according to Jaffe et al.14 Briefly, endothelial cells were isolated from umbilical veins by digestion with collagenase I for 15 min at 37°C. The vein was flushed with sterile medium to collect endothelial cells. The culture medium consisted M199 culture medium supplemented with 20% FBS, 2 mM L-glutamine, 100 U/mL penicillin,100 U/mL streptomycin, 40 µg/mL endothelial cell growth supplement (ECGS purchased from China-Japan Friendship Hospital, China) and 40 U/mL heparin. All experiments were performed with cells between passages 2 to 5. HUVECs were characterized by immunofluorescence for Von Willebrand factor and by microscopic observation of typical cobblestone morphology.

The cells were pretreated with 10–8–10–6 M Sal B for 24 h, after which the medium was removed and replaced with standard growth medium containing 500 µM H2O2. After additional 4 h incubation, the cells were evaluated as described later.

2.4 Cytotoxicity assays
Cell viability was measured by colorimetric assay with MTT. Briefly, cells were pretreated with various concentrations of Sal B for 24 h and followed by treatment with H2O2. After treatment, the cells were incubated with 0.5 mg/mL MTT at 37°C for 4 h, then the media was carefully removed and 100 µL of DMSO was added to dissolve the formed formazan product. The amount of MTT formazan product was determined by measuring optical density with a microplate reader (Thermo Fisher Scientific Inc., Waltham, MA, USA) at a wavelength of 570 nm and a reference wavelength of 655 nm.

Cell death was determined by measuring LDH activity. At the end of incubation, the supernatant was collected, and the content of LDH released from cells was determined using LDH assay kit according to the manufacturer’s instructions. The absorbance was measured on a microplate reader at 440 nm. The results were expressed as the percentage of the maximum amount of LDH released from samples that had been treated with 1% Triton X-100.

2.5 Multiparametric apoptosis assay by high content screening analyser
The Multiparamter Apoptosis 1 HitKit for HCS reagent kit was used to measure three fundamental parameters associated with the process of apoptosis: nuclear morphology, changes in F-actin content, and mitochondrial membrane potential (MMP). Nuclear morphology was observed after staining the cells with the fluorescent nuclear dye, Hoechst33342. MMP was determined based on the uptake of the fluorescent dye, MitoTracker Red, into the mitochondria of cells. The F-actin content was assayed by staining the cells with the fluorescent Alexa Fluor 488 Phalloidin. Plates with stained and fixed cells were analysed using ArrayScan HCS system (Cellomics Inc., Pittsburgh, PA, USA). Hundred cells were detected for each of the three microscopic fields per well.

2.6 Two-dimensional gel electrophoresis and image analysis
Monolayer HUVECs were treated with or without Sal B (10–6 M) for 24 h. Cells were harvested using homemade scrapers. The resulting pellets were resuspended in 100 µL of solubilization buffer consisting of 40 mM Tris, 8 M urea, 4% w/v CHAPS, 60 mM DTT and 1 mM PMSF. After three freeze-thaw cycles, samples were centrifuged at 13 000g for 10 min and supernatant was collected. First dimension isoelectric focusing (IEF) was performed using 7 cm pH 3–10 linear immobilized pH gradient readystrips (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Protein samples (120 µg) from each experimental group were solubilized in rehydration buffer, which contains 8 M urea, 4% CHAPS, 65 mM DTT, 0.2% Bio-Lyte (Bio-Rad) and 0.001% bromothymol blue, to a volume of 125 µL and allowed to incubate at room temperature for 30 min. After positive rehydration for 12 h at 50 V, IEF was run at 20°C in following steps: 250 V linear for 30 min, 500 V rapid for 30 min, 4000 V linear for 3 h, 4000 V rapid until 20 000 Vh. The IEF strips were then equilibrated by serial incubation (15 min) in equilibration buffer (6 M urea, 2% SDS, 0.375 M Tris–HCl (pH 8.8), 20% glycerol and 20 mg/mL DTT) and in equilibration buffer containing 2.5% iodoacetamide instead of DTT. Then, the samples were separated in the second dimension on 12% polyacrylamide gels at 80 V for 10 min and then at 110 V for 50–60 min. Gels were stained with Coomassie Blue. The differentially expressed protein spots were excised manually from the SDS–PAGE (sodium dodecyl sulphate–polyacrylamide gel) gels and subject to in-gel tryptic digestion.

2.7 Mass spectrometry identification of proteins
The protein of interest was analysed by nanoelectrospray with a hybrid quadrupole time-of-flight (Q-TOF) mass spectrometer (Waters, MA, USA). The peptide mixture was carried out on a Waters Capillary liquid chromatography system including three pumps A, B, and C (Waters). Fused silica tubing (75 µm x 100 mm) packed with symmetry 300TM C18, 3.5 µm spherical particles with a pore diameter of 100° (Waters). The flow rate was set at 2.5 µL/min. Samples were injected at a flow rate of 20 µL/min with pump C by the autosampler, and salts were removed on the precolumn of (0.35 mm x 5 mm) packed with symmetry 300TM C18, 3.5 µm spherical particles with a pore diameter of 100°. The Cap LC is coupled on-line with a Q-TOF ultima global mass spectrometer for detection and protein identification.

2.8 Ribonucleic acid isolation and reverse transcription–polymerase chain reaction
Total RNA was isolated using Trizol reagent (Invitrogen, Groningen, NL). First-strand cDNAs were generated from RNA samples by reverse transcription using oligo (dT). The following primers were used to amplify fragments of the human GRP78: 5'-TCCTATGTCGCCTTCACT-3' (sense) and 5'-ACAGACGGGTCATTCCAC-3' (antisense); ATF4: 5'-GTCTCCGTGAGCGTCCAT-3' (sense) and 5'-CAGAAGCCAACTCCCATTAG-3' (antisense); ATF6: 5'-CCCAAGACTCAAACAAAC-3' (sense) and 5'-TAATCTCGCCTCTAACCC-3' (antisense); β-actin: 5'-ATCATGTTTGAGACCTTCAACA-3' (sense) and 5'-CATCTCTTGCTCGAAGTCCA-3' (antisense).

2.9 Western blot analysis
Equal amounts of protein was subjected to SDS–PAGE on a 12% polyacrylamide gel and transferred to a polyvinylidene difluoride membrane (Millipore Corp, MA, USA). The membrane with blotted protein was blocked for 1 h with blocking buffer containing 5% non-fat dry milk and 0.05% Tween 20 in Tris-buffered saline (TBS-T), followed by incubation with indicated antibodies diluted 1:1000 in blocking buffer overnight at 4°C. Then, the membrane was washed three times with TBS-T for 30 min and incubated at room temperature for 1 h with diluted (1:2000) secondary horseradish peroxidase-conjugated goat anti-rabbit IgG (Santa Cruz, CA, USA). Detection was done using the ECL Western Blotting Detection System (Amersham Biosciences, Piscataway, NJ, USA).

2.10 Short interfering ribonucleic acid transfection
The short interfering ribonucleic acid (siRNA) duplexes used in this study were chemically synthesized by Genechem Corporation, Shanghai, China. 5'- CUGUUACAAUCAAGGUCUATdT-3' and 5'-UAGACCUUGAUUGUAACAGTdT or 5'-GGAGAUAGGAAGCCAGACUTdT-3' and 5'-AGUCUGGCUUCCUAUCUCCTdT-3' were used for repressing GRP78 or ATF4 expression, respectively. Non-silencing siRNA (5'-UUAAGUAGCUUGGCCUUGATdT-3' and 5'-UCAAGGCCAAGCUACUUAATdT -3') was used as a negative Control. siRNA duplexes were transfected into HUVECs with siRNA transfection reagent (Polyplus-transfection Inc., San Marcos, CA, USA) according to the manufacturer’s instructions.

2.11 Statistical analysis
Each experiment was performed at least three times and statistical analyses were performed using one-way ANOVA followed by Tukey’s test as a post hoc test with the SPSS software. The number of experimental samples used in each group is presented in the figure legends. All data are represented as the means ± SD and differences were considered significant at P < 0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Funding
 References
 
3.1 Salvianolic acid B protected human umbilical vein endothelial cell from H2O2-induced cytotoxicity
To evaluate whether Sal B protects HUVECs from oxidative stress, cell viability was determined using an MTT reduction assay. Cell survival was tested following treatment with a range of H2O2 concentrations (from 200 to 1 mM) and it was found that H2O2 significantly decreases cell survival in a dose-dependent manner. Cell viability ranged from 70.2 to 10.3%, respectively. In the presence of 500 µM of H2O2, there were 39.2 ± 1.7% (mean ± SEM, n = 3) viable cells compared with Control cells. Therefore, treatment with 500 µM H2O2 for 4 h was done in the subsequent experiments to provide a maximum dynamic range for quantifying protective vs. harmful responses. As illustrated in Figure 1C, Sal B (10–8, 10–7, 10–6 M) prevented H2O2-caused damage in the cells, restoring cell survival to 41.6 ± 4.6, 56.1 ± 2.7, and 78.8 ± 3.9% (mean ± SEM, n = 3), respectively.

To further investigate the protective effects of Sal B, the LDH assay was performed. As illustrated in Figure 1D, only 24.1 ± 2.5% of cellular LDH was released in the Control group. When HUVECs were exposed to H2O2 for 4 h, LDH leakage was markedly increased (65.1 ± 3.4%), while pretreatment of HUVECs with Sal B (10–8, 10–7, 10–6 M) reduced H2O2-induced LDH leakage in a concentration-dependent manner (64.3 ± 3.7, 49.7 ± 2.7, and 33.9 ± 3.6%, respectively).

3.2 Salvianolic acid B protected human umbilical vein endothelial cells against H2O2-induced apoptosis
The effects of Sal B on H2O2-induced apoptosis in HUVECs were analysed by three apoptosis parameters. Multiparameter fluorescent images and dose–response plots were obtained from the same microscopic field. Changes in the cell nucleus are observable using a fluorescent nuclear dye, Hochest33342. When HUVECs were stained with Hoechst33342, the normal HUVEC nuclei had lower than average fluorescence intensities. The cells that were treated with 500 µM H2O2 for 4 h showed typical characteristics of apoptosis, including an increase in the fluorescent intensity. However, in the Sal B-pretreated groups, the fluorescent intensities of Hoechst33258 were markedly decreased (Figure 2A).


Figure 2
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  		Figure 2 Protective effects of Salvianolic acid B (Sal B) on H2O2-induced apoptosis in human umbilical vein endothelial cells. Cells were pretreated with 10–8–10–6 M Sal B, respectively, followed by 500 µM H2O2 challenge for 4 h. Nuclear morphology was observed using Hoechst33342 staining. Mitochondrial membrane potential changes were based on the uptake of MitoTracker Red into the mitochondria of cells. The F-actin content was determined by the amount of fluorescent Alexa Fluor 488 Phalloidin staining. 100 cells were measured for each of the three microscopic fields-of-view per well. The average fluorescence intensities of Hochest33342 (A), MitoTracker Red (B), and Alexa Fluor 488 Phalloidin (C) were monitored by an HCS (high content screening) Reader. Values are expresses as the mean ± SD of three independent experiments, n = 6. ##P < 0.01, vs. H2O2-free group (Control); *P < 0.05, vs. H2O2 alone group; **P < 0.01, vs. H2O2 alone group.

 
The F-actin content is typically determined by the amount of fluorescent phalloidin staining. Alexa Fluor 488-Phalloidin binding is proportional to the amount of F-actin present. H2O2 resulted in a decrease in F-actin density. Sal B treatment attenuated the F-actin density decrease induced by H2O2 (Figure 2B).

Loss of MMP was quantifiable by MitoTracker Red. After incubation with 500 µM H2O2 for 4 h, the fluorescence intensity of MitoTracker Red was significantly decreased in HUVECs. Pretreatment with different concentrations of Sal B protected the cells from the H2O2-induced MMP lowering (Figure 2C).

3.3 Comparative proteomic analysis in human umbilical vein endothelial cells with or without Salvianolic acid B treatment
To elucidate novel molecular mechanisms by which Sal B protects endothelial cells from oxidative damage, we compared the protein expression patterns in Sal B- (10–6 M) treated and untreated cells. Two-dimensional gel electrophoresis (2-DE) analysis showed generally similar protein expression patterns in Sal B-treated and -untreated cells (Figure 3A). Five hundred and fifty one protein spots were detected in the untreated group and 447 protein spots were detected in the Sal B-treated group. Two obviously different spots (Figure 3B) were identified. The intensity of protein spot No. 1 significantly increased after Sal B treatment and the intensity of protein spot No. 2 decreased after Sal B treatment. Q-TOF analysis identified these proteins as GRP78 (Figure 3C) and histone H2B (Figure 3D), respectively. In this study, we predominantly focused on GRP78 because it plays an important role in protection against cytotoxicity and apoptosis.


Figure 3
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  		Figure 3 Two-dimensional gel electrophoresis (2-DE) analysis of protein expression in cells with or without Salvianolic acid B (Sal B) treatment. (A) Representative 2-DE maps of proteins in human umbilical vein endothelial cells without or with Sal B treatment. Approximately 120 µg of total protein was focused on linear immobilized pH gradient strips (pH 3–10, 7 cm) before separating on a 12% sodium dodecyl sulphate–polyacrylamide gel. Coomassie blue staining was used for detecting protein spots. (B) Close-up image of differential expression protein spots between cells with or without Sal B treatment. Two obviously different spots marked by arrows were selected for quadrupole time-of-flight mass spectrometer (Q-TOF-MS). (C) Q-TOF-MS analysis of protein No. 1 mass spectrometry of in-gel trypsin digests of this protein resulted in the identification of GRP78. (D) Q-TOF-MS analysis of protein No. 2 mass spectrometry of in-gel trypsin digests of this protein resulted in the identification of histone H2B.

 
3.4 Sal B up-regulated glucose-regulated protein 78 and glucose-regulated protein 94
Reverse transcription–polymerase chain reaction (RT–PCR) analysis was used to determine whether GRP78 transcription is regulated by Sal B. Increasing Sal B incubation time elevated GRP78 mRNA levels. GRP78 mRNA in HUVECs began to increase at 2 h and peaked at 6 h after treatment with 10–6 M Sal B (Figure 4A). The up-regulation of GRP78 was confirmed by western blot analysis. In HUVECs treated with 10–6 M Sal B, the level of GRP78 increased significantly at 6 h after treatment and remained at a high expression level for 24 h (Figure 4B). Sal B significantly increased GRP78 expression in a dose-dependent manner (Figure 4C). A time course of GRP78 transcription regulation nearly paralleled the effects on GRP78 protein expression regulation.


Figure 4
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  		Figure 4 Time course and dose effects of Salvianolic acid B (Sal B)-induced accumulation of glucose-regulated protein 78 (GRP78) and GRP94. (A) Time course of Sal B on GRP78 gene expression. Human umbilical vein endothelial cells (HUVECs) were grown for 0–24 h with 10–6 M Sal B. Control cells were treated identically, but without addition of Sal B. Total RNA (ribonucleic acid) was isolated and subjected to RT–PCR (reverse transcription–polymerase chain reaction) analysis using GRP78 and β-actin primers. (B) Time course of Sal B on GRP78 and GRP94 protein levels. HUVECs were incubated with 10–6 M Sal B for 2–24 h. (C) Dose effects of Sal B on GRP78 and GRP94 protein levels. Cells were treated with 10–9–10–5 M Sal B for 24 h. Cell lysates were analysed by western blotting using GRP78, GRP94, and β-actin antibodies. Autoradiographs were quantified by densitometry. Control values (Sal B untreated group) were set to one. Values are the mean ± SD of three experiments. *P < 0.05 vs. Control, **P < 0.01 vs. Control.

 
GRP94, which shares significant sequence homology with GRP78, was shown to have similar cytoprotective functions as GRP78. It is well established that GRP78 and GRP94 are regulated in a coordinated manner. Therefore, we examined the expression level of GRP94. The results indicated that the expression of GRP94 was also enhanced by Sal B (Figure 4B and C).

3.5 Mechanisms for up-regulation of glucose-regulated protein 78 by Salvianolic acid B
It is known that PERK signalling activates eIF2{alpha}, leading to increased expression of ATF4. ATF4 then binds to the promoter of the GRP78 gene thereby increasing GRP78 production.15 We examined whether Sal B could activate the PERK pathway in HUVECs. As shown in Figure 5B, Sal B (10–6 M) transiently increased the levels of p-PERK and p-eIF2{alpha}. Furthermore, up-regulation of ATF4 was confirmed by RT–PCR and immunoblotting analysis. Up-regulation of ATF4 mRNA was similar to that of GRP78 mRNA following Sal B treatment (Figure 5A). Interestingly, phosphorylation of PERK was detected within 2 h after the Sal B treatment, maximal expression of e-IF2{alpha} was reached 4 h after Sal B treatment and the peak level of ATF4 was observed at 24 h after addition. This observation is in agreement with the recent findings that the sequential activation of PERK, eIF2{alpha}, and ATF4 is involved in the up-regulation of GRP78.


Figure 5
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  		Figure 5 Salvianolic acid B (Sal B) activated PERK [pancreatic ER kinase (PKR)-like ER kinase], eIF2{alpha}, ATF (activating transcription factor) 4 and ATF6 in human umbilical vein endothelial cells (HUVECs). HUVECs incubated with 10–6 M Sal B for 0–24 h. (A) Time course of Sal B on mRNA (messenger ribonucleic acid) levels of ATF4. Total RNA was isolated and subjected to RT–PCR (reverse transcription–polymerase chain reaction) analysis using ATF4 and β-actin primers. (B) Activation of PERK [pancreatic ER kinase (PKR)-like ER kinase], eIF2{alpha} and ATF4 by Sal B. Cell lysates were subjected to western blotting using phosphorylated PERK (p-PERK), PERK, phosphorylated eIF2{alpha}, eIF2{alpha}, ATF4 and β-actin antibodies. (C) Effect of short interfering ribonucleic acid (siRNA) for ATF4 on the Sal B-induced glucose-regulated protein 78 (GRP78) up-regulation. HUVECs transfected without or with ATF4 siRNA or non-silencing (ns) siRNA were incubated with or without 10–6 M Sal B for 24 h. Western blot analysis shows ATF4 and GRP78 protein levels. (D) Time course of Sal B on mRNA levels of ATF6. Total RNA was isolated and subjected to RT–PCR analysis using ATF4 and β-actin primers. (E) Activation of ATF6 by Sal B. Cell lysates were subjected to western blotting using ATF6 antibody.

 
ATF4 siRNA was used to examine the contribution of this transcription factor to Sal B-dependent up-regulation of GRP78. Knockdown of ATF4 partially repressed Sal B-induced GRP78 expression (Figure 5C), suggesting that ATF4 is involved in Sal B-induced GRP78 up-regulation.

ATF6 is another factor that can constitutively induce GRP78 expression. ATF6 is a 90 kDa transmembrane glycoprotein (p90 ATF6) embedded in the ER under normal growth conditions. In response to ER stress, endogenous ATF6 is cleaved into a 50 kDa soluble nuclear protein (p50 ATF6) to induce GRP78 expression.16 We first used RT–PCR techniques to test the effect of Sal B on ATF6 mRNA expression. As shown in Figure 5D, ATF6 mRNA levels increased after 2 h Sal B treatment, and this elevation became more pronounced for various times. The effect of Sal B on the activation of ATF6 was also analysed by western blot analysis. When HUVECs were treated with Sal B, p90 ATF6 was detected in a sustained manner in comparison with p50 ATF6, which was only transiently detected (Figure 5E). These data further suggested that ATF6 was activated by Sal B.

3.6 Effects of glucose-regulated protein 78 knockdown on the cytoprotection of Salvianolic acid B
SiRNA was used to confirm that Sal B-induced GRP78 protects cells from H2O2-induced cell injury. All of the GRP78 siRNA-treated groups showed lower levels of protein as compared with their counterparts (Figure 6A). Transfection with GRP78 siRNA significantly decreased the cell viability after H2O2 treatment either with, or without Sal B pretreatment. The protective effect of Sal B was mostly counteracted by the GRP78 siRNA. The scrambled negative Control siRNA did not alter cell viability (Figure 6B). These results strongly suggest that Sal B promotes HUVEC cell survival through up-regulation of GRP78 expression.


Figure 6
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  		Figure 6 Glucose-regulated protein 78 (GRP78) knockdown partially inhibited Salvianolic acid B (Sal B)-induced improvement in human umbilical vein endothelial cells (HUVECs) survival under oxidative stress. HUVECs were transfected with or without GRP78 short interfering ribonucleic acid (siRNA) for 24 h, then incubated with or without 10–6 M Sal B for 24 h, followed by 500 µM H2O2 for another 4 h. (A) Western blot analysis of GRP78 protein levels. Total protein of each sample was subjected to western blot with GRP78 and β-actin antibody. (B) Cell viability was measured in HUVECs transfected with or without siRNA against the GRP78 (Neg C, non-silencing siRNA). Values are the mean ± SD of three experiments, n = 6; *P < 0.05; **P < 0.01.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Funding
 References
 
Many vascular functions are redox-sensitive processes, and the basis of vascular health lies in the delicate balance between ROS-producing and ROS-detoxifying systems. Overproduction of ROS is one of the important contributors in the development of cardiovascular diseases.2 Thus, removal of excess ROS or suppression of ROS generation by antioxidants may be effective in preventing oxidative stress-induced cell injury. Considerable efforts have been devoted to searching for natural antioxidants that impart cardiovascular benefits. Sal B, a major compound occurring naturally in Danshen, was reported to possess strong antioxidant activities.17 The purpose of this study was to evaluate whether Sal B protects endothelial cells from oxidative damage and to identify the possible protective molecular mechanisms of this compound.

We established an in vitro oxidative injury model using H2O2 to induce oxidative stress in the vascular lumen. The exposure of cultured cells to H2O2 results in injury to lipids, proteins and nucleic acids. This damage may lead to vascular structural and functional impairments.18 Here we demonstrated that H2O2 treatment increased LDH leakage and decreased the viability of HUVECs. Pretreatment with Sal B (10–8–10–6M) significantly ameliorated the LDH leakage and preserved cell viability of HUVECs. These results indicate that Sal B protects HUVECs from H2O2-induced cytotoxicity. The concentrations of Sal B used in this study were designed to be relevant to the pharmacokinetics of Sal B in humans. Previous reports showed that after administration of Danshen injection fluid, the concentrations of Sal B in human serum ranged from 21.3 to 1518 ng/mL (2.9 x 10–8 to 2.1 x 10–6 M), which are within the range that we used in the present study.19 Preliminary experiments were also performed to determine the duration of Sal B exposure to HUVECs. We used different dosages of Sal B (10–9–10–5 M) to treat HUVECs for various times (2–48 h). The cells were then exposed to 500 µM H2O2 for 4 h. The results suggest that after a 24 h incubation period, Sal B exerts the strongest protective effects. Therefore, a 24 h treatment with Sal B was used in the subsequent experiments.

Next, we explored whether Sal B protected HUVECs from apoptosis. HCS is a new technique combining quantitative epifluorescence microscopy and automatic image analysis.20 Our assay generated data related to three important hallmarks of apoptosis. These include the disruption of MMP, changes in F-actin and nuclear fragmentation. ROS-induced apoptosis often involves the loss of the MMP leading to the subsequent release of apoptosis-inducing factors from the mitochondria. The released factors eventually lead to nuclear condensation and fragmentation.21 Our present study shows that H2O2 induced a profound loss of MMP, while Sal B preserved the MMP in H2O2-exposed cells in a concentration-dependent manner. H2O2-treated cells displayed typical morphological features of apoptosis. Sal B pretreatment lessened these nuclear alterations. In addition to disruption of the MMP and nuclear fragmentation, the cytoskeletal network is also one of the earliest targets of oxidative stress. ROS can induce cell morphology changes and F-actin reorganization in endothelial cells.22 Our results showed that H2O2 induced the reduction of the fluorescent intensity of F-actin bands. However, pretreatment with Sal B attenuated the reorganization of F-actin filaments induced by H2O2. Taken together, these data strongly suggest that Sal B protects HUVECs from H2O2-induced apoptosis.

With a phenolic hydroxyl group in its molecular structure, Sal B displays a strong free radical scavenging ability that may partially account for its anti-apoptotic effects.23 However, the possibility that Sal B provides its protective effects via other mechanisms cannot be excluded. A growing body of evidence indicates that protein regulations are important events for proper endothelial cell function.24 Thus, we used proteomic tools to investigate the mechanism underlying Sal B protective activity.

The proteomic analysis revealed that GRP78 expression increased in response to Sal B treatment. GRP78 is a chaperone protein and a central regulator of endoplasmic reticulum (ER) function and because of these roles, it is important in many cellular processes. These processes include protein folding, protein assembly, targeting misfolded proteins for degradation, ER Ca2+-binding and controlling the activation of trans-membrane ER stress sensors.25 Induction of GRP78 appears to maintain cellular homeostasis and prevent cells from apoptosis.26 Due to its anti-apoptotic properties, induction of GRP78 represents an important prosurvival component of ER stress. It has been reported that, prior to ER stress, induced GRP78 aggregation protects cells against a subsequent challenge with a variety of oxidative insults.27,28 Conversely, knockdown of GRP78 sensitizes cells to oxidative stress.29,30 We therefore hypothesized that GRP78 may mediate the protective effects of Sal B.

The present investigation showed that treatment with Sal B induced the accumulation of GRP78 in a time- and dose-dependent manner in HUVECs. Also the dose–response curve for the up-regulation of GRP78 by Sal B was related to the protection from cell death. Transcriptional up-regulation of GRP78 is the hallmark of the ER stress response.25 This suggested Sal B might up-regulate GRP78 by exerting ER stress. Therefore, we also examined the effect of Sal B on GRP94, another marker of the ER stress.31 Our data indicated that Sal B also up-regulated GRP94. Several studies reported a significant increase in GRP94 levels in different cell types after exposure to mild ischemia and to other conditions known to trigger delayed cytoprotection.32,33 Other elegant study demonstrated that reduced levels of GRP94 was accompanied by reduced cell viability.34 Therefore, induction of GRP94 may be another important mechanism for the protective effects of Sal B. The simultaneous induction of GRP78 and GRP94 suggest that Sal B pretreated cells under ER stress.

In order to further confirm that Sal B-induced GRP78 expression protects cells from H2O2-induced cell injury, we used siRNA to down-regulate GRP78 expression in HUVECS. Our data showed that GRP78 knockdown cells were more sensitive to H2O2-induced toxicity than cells without siRNA transfection. This indicated that knocking down GRP78 significantly reduced the protective effects of Sal B. Our results are consistent with reports that cells expressing an antisense GRP78 construct are more susceptible to cellular damage induced by hydrogen peroxide,35 iodoacetamide,30 and tert-butylhydroperoxide.27 Taken as a whole, these data suggest that the stimulatory effects of Sal B on GRP78 are critical for Sal B-mediated cytoprotection.

The type-I ER transmembrane protein kinase (IRE1), the eukaryotic translation initiation factor 2 kinase (PERK) and the ATF6 are three ER-localized sensor proteins involved in the transcriptional activation of the GRP78 promoter.36 We found when HUVECs were treated with Sal B, ATF4 expression was markedly increased and phosphorylation of eIF2{alpha} protein occurred in advance to the accumulation of ATF4 protein. ATF4 siRNA partially inhibited the Sal B-induced GRP78 changes, indicating there are other pathways except the PERK–eIF2{alpha}–ATF4 pathway involved in this mechanism. ATF6 is a basic leucine zipper protein that binds to the consensus ER stress response element and enhances the transcription of genes encoding GRP78. In response to ER stress, ATF6 translocates from the ER to the Golgi, where it is cleaved. The cleaved portion of ATF6 then enters the nucleus as a transcription factor for GRP78. Our results indicated that ATF6 was activated when exposed to Sal B. Therefore, both ATF6 and PERK–eIF2{alpha}–ATF4 pathways are involved in Sal B-induced GRP78 up-regulation.

In conclusion, this study demonstrated for the first time that Sal B protects endothelial cells against oxidative stress-induced cell injury via up-regulating GRP78. This is a new mechanism that may be unrelated to previously known direct free radical-scavenging activities. Sal B and Sal B-containing herbal medicines could be useful clinically as GRP78 inducers. Further investigation, including animal modeling, is required to elucidate the protective role, or roles, of GRP78 expression as induced by Sal B under in vivo conditions of oxidative stress-induced vascular damage.


   Funding
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
References
 
This work was supported by the National Natural Science Foundation of China (No. 30171090, 30270528, 30572202, 30772571), 973 Program of the Ministry of Science and Technology in China (No. 2004CB518902), research fund from Ministry of Education of China (111 Projects No. B0700 and 985 Project).


   Acknowledgements
 
The authors are grateful to Professor Bao-Xue Yang (Department of Pharmacology, School of Basic Medical Sciences, Peking University) and Joel Lechner (Department of Biochemistry, University of Nebraska-Lincoln) for a critical reading of the manuscript.

Conflict of interest: none declared.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
 References
 

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