Cardio-protection of salvianolic acid B through inhibition of apoptosis network.

Abstract

Targeting cellular function as a system rather than on the level of the single target significantly increases therapeutic potency. In the present study, we detect the target pathway of salvianolic acid B (SalB) in vivo. Acute myocardial infarction (AMI) was induced in rats followed by the treatment with 10 mg/kg SalB. Hemodynamic detection and pathological stain, 2-dimensional electrophoresis, MALDI-TOF MS/MS, Western blot, pathway identification, apoptosis assay and transmission electron microscope were used to elucidate the effects and mechanism of SalB on cardioprotection. Higher SalB concentration was found in ischemic area compared to no-ischemic area of heart, correlating with improved heart function and histological structure. Thirty-three proteins regulated by SalB in AMI rats were identified by biochemical analysis and were classified as the components of metabolism and apoptosis networks. SalB protected cardiomyocytes from apoptosis, inhibited poly (ADP-ribose) polymerase-1 pathway, and improved the integrity of mitochondrial and nucleus of heart tissue during AMI. Furthermore, the protective effects of SalB against apoptosis were verified in H9c2 cells. Our results provide evidence that SalB regulates multi-targets involved in the apoptosis pathway during AMI and therefore may be a candidate for novel therapeutics of heart diseases.

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Cardio-Protection of Salvianolic Acid B through
Inhibition of Apoptosis Network
Lingling Xu1, Yanping Deng1, Lixin Feng1, Defang Li1, Xiaoyan Chen1, Chao Ma1, Xuan Liu1, Jun Yin2,
Min Yang1, Fukang Teng1,2, Wanying Wu1, Shuhong Guan1, Baohong Jiang1*, Dean Guo1*
1Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China, 2Shenyang Pharmaceutical University, Shenyang, China
Abstract
Targeting cellular function as a system rather than on the level of the single target significantly increases therapeutic
potency. In the present study, we detect the target pathway of salvianolic acid B (SalB) in vivo. Acute myocardial infarction
(AMI) was induced in rats followed by the treatment with 10 mg/kg SalB. Hemodynamic detection and pathological stain, 2-
dimensional electrophoresis, MALDI-TOF MS/MS, Western blot, pathway identification, apoptosis assay and transmission
electron microscope were used to elucidate the effects and mechanism of SalB on cardioprotection. Higher SalB
concentration was found in ischemic area compared to no-ischemic area of heart, correlating with improved heart function
and histological structure. Thirty-three proteins regulated by SalB in AMI rats were identified by biochemical analysis and
were classified as the components of metabolism and apoptosis networks. SalB protected cardiomyocytes from apoptosis,
inhibited poly (ADP-ribose) polymerase-1 pathway, and improved the integrity of mitochondrial and nucleus of heart tissue
during AMI. Furthermore, the protective effects of SalB against apoptosis were verified in H9c2 cells. Our results provide
evidence that SalB regulates multi-targets involved in the apoptosis pathway during AMI and therefore may be a candidate
for novel therapeutics of heart diseases.
Citation: Xu L, Deng Y, Feng L, Li D, Chen X, et al. (2011) Cardio-Protection of Salvianolic Acid B through Inhibition of Apoptosis Network. PLoS ONE 6(9): e24036.
doi:10.1371/journal.pone.0024036
Editor: Carlo Gaetano, Istituto Dermopatico dell’Immacolata, Italy
Received February 25, 2011; Accepted July 29, 2011; Published September 6, 2011
Copyright: ? 2011 Xu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the National Science and Technology Major Project for ‘‘Key New Drug Creation and Manufacturing Program’’
(2009ZX09308-005; 2009ZX09102-122; 2009ZX090304-002; 2009ZX09311-001; 2009ZX09502-020). The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: daguo@mail.shcnc.ac.cn (DAG); jiangbh@mail.shcnc.ac.cn (BHJ)
Introduction
Traditionally, the goal in drug development is to design selective
compounds that act on single disease target to achieve desired
potency with low side effects. This strategy of ‘‘one-gene, one-
drug, one-disease’’ turns out to be unsuccessful for the majority of
‘‘complex disease’’ because pathogenesis of many diseases is multi-
factorial rather than due to a single cause [1]. Cardiovascular
diseases, the leading cause of mortality and morbidity in the world,
are one class of such ‘‘complex diseases’’. Many cardiovascular
diseases are the results of relatively subtle dysfunctions of multi
physiological pathways [2]. Development of new compounds
targeting different pathways and acting as multi-target inhibitors
provides new strategies for cardiovascular therapy. Such poly-
pharmacological approach to target cellular function as a whole
rather than each individual target within the pathway may
increase therapeutic potency [3].
Salvia miltiorrhiza has been widely used in clinic in China for the
treatment of various microcirculatory disturbance-related diseases,
such as cardiovascular disease, cerebrovascular disease, renal
dysfunction, liver fibrosis, and diabetic vascular complication [4].
Salvianolic acid B (SalB) is the major water-soluble component
extracted from Salviae miltiorrhizae. Several lines of in vitro
experiments have demonstrated biological activities of SalB in
promoting cellular proliferation and differentiation, anti-apoptotic,
preserving normal cellular functions [5–9]. Our recent data
showed that SalB protects against cardiac remodeling through
inhibition of matrix metalloproteinase-9 and fibrosis [10]. Base on
these reports, SalB seems to have pleiotropic effects and may act
on multiple molecular targets, making it a suitable candidate for
polypharmacology strategy to develop novel cardiovascular
therapeutics.
In the present study, we used myocardial infarction as a disease
model to evaluate protective effect of SalB on diseased heart. A
series of assays were employed to explore the potential mechanism
of SalB cardioprotection including biochemical, cardiophysiologi-
cal, histopathological, and pathway analysis. Our results indicated
that SalB regulated multi-targets involved in the apoptosis
pathway during acute myocardial infarction and therefore might
be a candidate for further polypharmacology research on novel
therapeutics of cardiovascular diseases.
Materials and Methods
Animal model and SalB treatment
Wistar male rats (230–250 g) were purchased from Shanghai
Center of Experimental Animals, Chinese Academy of Sciences.
The purity of SalB (purchased from Shanghai Yousi Bio-Tech
Co., Ltd.) was more than 99% evaluated by high-performance
liquid chromatography (Figure S1) and the chemical structure of
SalB was elucidated by1H NMR and13C NMR (Figure S2, S3,
S4). AMI was introduced by ligation of the left anterior descending
coronary artery near the main pulmonary artery as described
previously [11]. Animals were randomly assigned into four groups:
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sham operated rats given saline (Sham, n=30), sham operated rats
given SalB (Sham-SalB, n=40), AMI rats given saline (AMI,
n=30), AMI rats given SalB (AMI-SalB, n=40). Thirty min later
and 24 h later after surgery, saline or SalB was administered
through intravenous (IV) injection (10 mg/kg). The dose of
10 mg/kg was set according the protection of SalB on ischemic
area (Figure S5). Ten out of 40 rats from Sham-SalB or AMI-SalB
were used to measure tissue distribution of SalB. This study was
carried out in strict accordance with the recommendations in the
Guide for the Care and Use of Laboratory Animals of National
Institutes of Health. All protocol was approved by Institutional
Animal Care and Use Committee at Shanghai Institute of Materia
Medica (IACUC number: SIMM-AE-GDA-2010-03).
Tissue distribution of SalB
Tissue-distribution of SalB in Sham rats or AMI rats was
detected. One-hundred-fifty mg of each tissue sample was
homogenized in methanol for approximately 1 min (1:5 ration
of tissue to methanol) using a Fluko F6/10 superfine homogenizer.
Ultrasonic treatment was applied for 5 min, and then the resulting
samples were centrifuged at 3500 rpm for 5 min at 4uC.
Supernatant was used to detect SalB concentration by chroma-
tography (Agilent Waldbronn, Germany). An API 4000 triple
quadrupole mass spectrometer equipped with a TurboIon Spray
ionization interface (Applied Biosystems, Canada) was used for
mass analysis and detection. Data acquisition was performed with
Analyst 1.4.1 software (Applied Biosystems, USA).
Measurements of hemodynamic parameters and cardiac
output
Twenty-five hours after surgery, the rats were anesthetized, and
a Mikro-tipped SPR-320 catheter (Millar Instruments Inc) was
inserted through the right carotid artery into left ventricle. Heart
rate, mean arterial pressure (MAP), left ventricular systolic
pressure (LVSP), end-diastolic pressure (EDP) of rats were
recorded by PowerLab 8/30 instrument (ADInstruments, Aus-
tralia). Maximal rate of pressure development for contraction
(+dP/dtmax) and maximal rate of pressure development for
relaxation (2dP/dtmax) were all calculated from the continuously
collected pressure signal. For cardiac output detection, rats were
anaesthetized, placed on a heating pad supplied with ventilation.
The thymus lobes were pulled apart to expose the aorta. The
ascending aorta was dissected and a transonic perivascular MA2.5
PSL flow probe (Transonic Systems, USA) was positioned around
the aorta. Appropriate amount of ultrasound transmission gel was
injected into the space between the probe and the aorta. Flow
signals were then acquired by TS420 flowmeter and PowerLab
recording unit.
Histopathological detection
After detection of hemodynamic parameters, the heart samples
were fixed by 4% neutral-buffered paraformaldehyde for 24 h,
and the specimens were paraffin-embedded, cut at 5 mm and were
stained with haematoxylin and eosin. Photomicrographs were
taken using an Olympus BX51 microscope plus Olympus DP71
CCD camera (Olympus Corporation, Japan).
Two-dimensional electrophoresis
Heart tissues from rat infarct area were pulverized under liquid
nitrogen and then detergent soluble proteins were extracted by
incubation in lysis buffer. Homogenization was achieved by
ultrasonication on ice, then centrifugation at 15, 0006g for 30 min
at 4uC. The supernatants were used for two-dimensional
electrophoresis as described previously using Bio-Rad 2-DE
system [12]. Briefly, 150 mg protein sample was applied for IEF
using the ReadyStrip IPG Strips. The strips were placed into a
Protein IEF cell and were rehydrated at 50 V for 12 h and then
the proteins were separated based on their pI. After IEF, the IPG
strips were equilibrated for 15 min in a buffer containing 50 mM
Tris-HCL, pH 8.8, 30% glycerol; 7 M urea, 2% SDS and 1%
DTT, and then applied on to 12% homogeneous SDS-PAGE gels
for electrophoresis using a PROTEIN II xi Cell system (Bio-Rad,
USA). The gels were then silver stained using Bio-Rad Silver Stain
Plus kit according to the manufacturer’s instructions. For each
protein sample, three independent electrophoreses were per-
formed to ensure reproducibility.
Image analysis and MALDI-TOF MS/MS
The silver-stained gels were scanned using a Densitometer GS-
800 (Bio-Rad, USA) and the images were analyzed using PD-
Quest software (Bio-Rad, USA). Proteins of interest were excised
from the gels with EXQuest Spot Cutter (Bio-Rad, USA), and MS
analysis was performed as described previously [13]. Briefly, gel
pieces were destained for 2 min, washed twice with deionized
water and shrunk by dehydration in ACN. The samples were then
swollen in a digestion buffer containing 25 mM ammonium
bicarbonate and 12.5 ng/ml trypsin at 4uC. After a 30 min
incubation, the gels were digested for over 12 h at 37uC. Peptides
were extracted twice using 0.1% TFA in 50% ACN, and the
extracts were dried under N2. For MALDI-TOF MS/MS,
peptides were mixed with 0.7 ml MALDI matrix and spotted on
the 192-well stainless steel MALDI target plates. MS measure-
ments were carried out on an ABI 4700 Proteomics Analyzer with
delayed ion extraction (Applied Biosystems). Data search files were
generated and then the database search was performed by using
the MASCOT search engine (Matrix Science, United Kingdom).
Protein homology identifications of the top hit (first rank) with a
relative score exceeding 95% probability and additional hits
(second rank or more) with a relative score exceeding 98%
probability threshold were retained. Proteins with protein score
more than 62 and best ion score more than 30 were accepted.
Pathway identification
KEGG was used to analysis all differential expressed proteins in
2-DE experiment and a list of target pathways was generated [14].
Figure 1. Accumulation of SalB in ischemic area of heart. All the
values are expressed as mean 6 S.E. ***p,0.001 versus no-infarct area
of Sham. n=10 for every group.
doi:10.1371/journal.pone.0024036.g001
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To explore the biological relevance underlying these target
pathways, linked pathways with these target pathways were also
retrieved from KEGG database and the pathway networks were
constructed using Pajek network visualization software (version
1.25).
Western blot analysis
Western blot analysis was performed on the infarct samples
using antibodies against the following proteins purchased from
Cell Signaling: poly(ADP-ribose) polymerase-1 (PARP-1), cleaved
PARP-1, IKKa, IKKb, p-IKKa, NF-kB, GAPDH. Antibody for
L-lactate dehydrogenase B (LDHB) was purchased from Abcam
and b-actin was from Beyotime. The aliquots of 25 mg protein
were electrophoresed on a SDS–PAGE gel and transferred to
polyvinylidene difluoride membrane. The membrane was blocked
for 1 h in blocking buffer containing 5% non-fat dry milk and
0.07% Tween 20 in Tris-buffered saline (TBS-T), followed by
incubation with indicated antibodies in blocking buffer overnight
at 4uC. Then, the membrane was washed three times with TBS-T
for 30 min and incubated at room temperature for 1 h with
secondary antibody. Detection was done using the ECL Western
Blotting Detection System (Pierce, USA). The bands density for
corresponding protein was quantified using a MiniBis system
(DNR Bio-Imaging Systems Ltd, Israel).
TUNEL assays
To evaluate apoptosis in infarct area of rat heart, terminal
deoxynucleotide transferase-mediated dUTP nick-end labeling
(TUNEL) assay using FragELTMDNA Fragmentation Detection
Kit was carried out according to the manufacturer’s instructions
(Calbiochem, USA). Briefly, the heart samples were fixed by
incubating in 4% neutral-buffered paraformaldehyde for 24 h,
Figure 2. SalB improves cardiac function of AMI rats. Mean arterial pressure (MAP), maximum rate of pressure development for relaxation
(2dP/dtmax), left ventricular systolic pressure (LVSP), maximum rate of pressure development for contraction (+dP/dtmax), end-diastolic pressure
(EDP), and cardiac output were demonstrated. All the values are expressed as mean 6 S.E. *p,0.05, ***p,0.001 versus Sham; #p,0.05, ##p,0.01
versus AMI. n=30 for every group.
doi:10.1371/journal.pone.0024036.g002
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and the specimens were then paraffin-embedded, cut to 5 mm
slices. The sections were de-paraffinized, rehydrated, and
permeabilized by incubation in solutions containing proteinase
K. After equilibration and labeling reaction, the results were
evaluated using BX51 fluorescence microscope plus Olympus
DP71 CCD camera (Olympus Corporation, Japan). Total cells
were visualized at 330–380 nm for DAPI staining, and apoptotic
cells were visualized at 465–495 nm for FITC staining.
Transmission electron microscopy
Hearts were dissected and small pieces from ischemic area were
cut and fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer
for 2 h, post-fixed in 1% osmium tetroxide in 0.1 M cacodylate
buffer for 1 h and embedded as monolayers in LX-112 (Ladd
Research Industries, USA). Ultrathin sections were cut and
contrasted with uranyl acetate followed by lead citrate and
observed with a Tecnai 12 BioTwin transmission electron
microscope (Philips Electronic Instruments, USA). Random
sections were selected for analysis by an electron microscopy
technician blinded to the treatments.
Cell culture and hypoxia challenge
Rat H9c2 cells (Cell bank of Chinese Academy of Sciences,
Shanghai) were maintained in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 10% fetal calf serum and
cultured in 5% CO2at 37uC. All media and culture reagents were
products of Gibco (Grand Island, NY) unless specified otherwise.
Just before induction of hypoxia challenge, vehicle or SalB was
added into the serum free culture media and the cells were placed
into a humidified hypoxia incubator (Tri-Gas Incubator, Heal
Force) to achieve a low-oxygen environment (95% N2 and 5%
CO2) for 24 h. Mitochondrial membrane potential detection and
DAPI stain were performed following.
Mitochondrial membrane potential detection using JC-1
A fluorescent cationic dye, 5,59, 6,69-tetrachloro-1,19, 3,39-
tetraethylbenzimidazolyl-carbocyanine iodide, commonly known
as JC-1 (Beyotime), was used to measure mitochondrial membrane
potential. After hypoxia treatment, cells grown on cover slips were
washed twice with PBS and incubated with 5 mg/ml JC-1 dye in
serum-free medium for 20 min at 37uC. Cells were washed three
times with PBS and analyzed immediately under a fluorescent
microscope after mounting with DAKO mounting medium. JC-1
fluorescence was measured from single excitation (488 nm) with
dual emission (shift from green 530 nm to red 590 nm). Green
fluorescence reveals the monomeric form of the JC-1 molecule,
which appears in the cytosol after mitochondrial membrane
depolarization. Red fluorescence is emitted from the aggregation
of JC-1 molecules, which is formed in the inner membrane of
actively respiring mitochondria. Photomicrographs were taken
using an Olympus BX51 microscope plus Olympus DP71 CCD
camera (Olympus Corporation, Japan).
Apoptosis detection by staining of nuclei with DAPI
The nuclei of H9c2 cells were stained with chromatin dye DAPI
(Invitrogen, USA). Briefly, after treatment, the cells were fixed
with 4% paraformaldehyde for 20 min, washed twice with PBS,
and incubated with 10 mg/ml DAPI in PBS at room temperature
for 30 min. After three washes, the cells were observed under an
Olympus fluorescence microscope (BX51) plus Olympus DP71
CCD camera. Total cells and apoptosis cells per field were
counted by a blinded investigator. The quantitative data was
expressed as percentage of apoptosis cell per field.
Statistical analysis
All quantitative data are reported as mean 6 S.E. Statistical
analyses were performed using one-way ANOVA to compare
means, followed by post hoc test. p,0.05 was considered to be
statistically significant.
Results
Accumulation of SalB in the ischemic area of heart
First we analyzed SalB concentration in several major organs.
1 h after intravenous administration of 10 mg/kg SalB, major
Figure 3. SalB improves heart structure of AMI rat detected by H&E stain. Representative photomicrographs from heart tissue of Sham,
Sham-SalB, AMI, AMI-SalB groups were shown. (A) 1006magnification. (B) 4006magnification. n=10 for each group.
doi:10.1371/journal.pone.0024036.g003
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organs (heart, kidney, and lung) were harvested and tissue
homogenate was prepared. The concentration of SalB in heart is
60.866.7 ng/g (no-infarct area of Sham); 79.2612.8 (no-infarct
area of AMI); 65.169.2 ng/g (infarct area of Sham) and
928.26135.8 ng/g (infarct area of AMI), respectively. There was
no difference of SalB concentration in liver and kidney between
Sham and AMI rats, the concentrations were all below 50 ng/g
(Figure 1). The higher concentration of SalB at ischemic area of
heart promoted us to concentrate this area for further mechanism
research.
SalB improves left ventricle function of AMI rat
To evaluate the possible protective effect of higher concentration of
SalB in heart of AMI rats, we measured several major haemodynamic
parameters for left ventricle contractility, including MAP, 2dp/dtmax,
LVSP, +dp/dtmax, and EDP along with cardiac output (Figure 2).
Compared to rats in the Sham group, left ventricle dysfunction in
AMI rats was observed with significant decrease of 2dp/dtmax
(25153.26257.4 mmHgS21versus29578.06546.0 mmHgS21,p,
0.001), LVSP (96.762.5 mm Hg versus 121.461.8 mm Hg,
p,0.001), +dp/dtmax (6134.16441.6 mmHgS21versus 9184.66
Table 1. Statistical analysis of 2-DE purified proteins changing in abundance following 24 h MI.
Spot no.
PPM
ShamSham-SalBAMIAMI-SalB
10.03160.0600.04760.08077.44620.41**43.53631.79#
20.03160.0600.04760.080129.23651.58** 64.88637.44#
365.95634.70108.58657.93178.07664.22**375.036222.66#
40.01760.0391.6264.2821.97630.42*0.0460.052#
560.05619.9681.72661.2495.14620.92**59.20629.08##
610195.0764565.2711004.5164404.934084.7162940.35**1999.196585.55#
70.03160.060297.376220.76**552.396297.49**242.35677.83#
8 5123.7962121.375493.8662338.043523.4961814.43*1925.076575.30##
9608.766815.451061.7161353.482982.8662114.23**962.096595.63#
101511.336607.93792.08679.81**687.976409.98**414.326170.65#
11106.34676.43154.806107.02210.646121.70*103.03633.22#
120.01560.040.04060.0854.89629.99**0.05460.07##
13129.39654.38135.09686.4068.23628.87**37.35631.95#
14781.386250.70561.726180.01**292.166170.23**139.26641.86##
1542.92634.0233.32623.700.09360.08**17.19610.94**
1653.20629.9948.99629.58104.55647.66**64.22630.20#
17241.556115.54269.826123.72446.346199.24**323.656130.49
18391.196237.48346.716211.541057.866403.52**1451.146435.50#
191687.696845.211428.556845.21310.116149.48**189.72667.31#
2073.58636.7524.07665.91*159.28675.45**247.92694.95#
2183.01667.88 118.02690.42296.846244.00**166.416158.52
22245.956110.24180.276104.2998.27644.93**50.50616.83##
23113.45671.38113.25671.12499.886221.35**834.656241.87##
24152.40677.4073.01683.24**87.38650.26* 37.85623.21##
25407.246257.81447.066306.43210.85666.85** 129.36665.89##
26 1443.746522.571175.326553.83311.306141.47**173.95659.41##
2760.58656.2561.02653.62105.84655.73* 39.78621.18##
28218.34696.84162.44679.81139.34652.49* 98.96625.96#
29 186.22673.60147.78665.38 84.43626.32**54.68634.47#
30 1407.176653.521545.666773.55 393.716255.81**222.77698.22#
3177.76645.5568.01639.4133.33617.79**15.12610.97##
32341.836200.10260.626129.4477.42671.56**23.63622.87#
33303.746182.15555.256633.07172.836104.76*63.01635.91##
*p,0.05,
**p,0.01,
***p,0.001 versus Sham;
#p,0.05,
##p,0.01,
###p,0.001 versus AMI.
doi:10.1371/journal.pone.0024036.t001
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