Cardioprotection of CAPE-oNO2 against myocardial ischemia/reperfusion induced ROS generation via regulating the SIRT1/eNOS/NF-κB pathway in vivo and in vitro

Article abstract of DOI:10.1016/j.redox.2017.11.023

Caffeic acid phenethyl ester (CAPE) could ameliorate myocardial ischemia/reperfusion injury (MIRI) by various mechanisms, but there hadn't been any reports on that CAPE could regulate silent information regulator 1 (SIRT1) and endothelial nitric oxide syn

Full text of DOI:10.1016/j.redox.2017.11.023

RedoxBiology15(2018)62–73
Contents lists available at ScienceDirect
Redox Biology
journal homepage: www.elsevier.com/locate/redox
Cardioprotection of CAPE-oNO2 against myocardial ischemia/reperfusion
induced ROS generation via regulating the SIRT1/eNOS/NF-κB pathway in
vivo and in vitro
Dejuan Lia,1, Xiaoling Wanga,1, Qin Huanga, Sai Lia, You Zhoub, Zhubo Lia,⁎
a
College of Pharmaceutical Sciences, Southwest University, No. 2, Tiansheng Road Beibei, Chongqing 400716, PR China
College of Biotechnology, Southwest University, Chongqing 400716, PR China
b
A R T I C L E I N F O
A B S T R A C T
Keywords:
Caffeic acid phenethyl ester (CAPE) could ameliorate myocardial ischemia/reperfusion injury (MIRI) by various
mechanisms, but there hadn’t been any reports on that CAPE could regulate silent information regulator 1
(SIRT1) and endothelial nitric oxide synthase (eNOS) to exert cardioprotective effect. The present study aimed to
investigate the cardioprotective potential of caffeic acid o-nitro phenethyl ester (CAPE-oNO2) on MIRI and the
possible mechanism based on the positive control of CAPE. The SD rats were subjected to left coronary artery
ischemia /reperfusion (IR) and the H9c2 cell cultured in hypoxia/reoxygenation (HR) to induce the MIRI model.
Prior to the procedure, vehicle, CAPE or CAPE-oNO2 were treated in the absence or presence of a SIRT1 inhibitor
nicotinamide (NAM) and an eNOS inhibitor Nω-nitro-L-arginine methyl ester (L-NAME). In vivo, CAPE and CAPE-
oNO2 conferred a cardioprotective effect as shown by reduced myocardial infarct size, cardiac marker enzymes
and structural abnormalities. From immunohistochemical and sirius red staining, above two compounds ame-
liorated the TNF-α release and collagen deposition of IR rat hearts. They could agitate SIRT1 and eNOS ex-
pression, and consequently enhance NO release and suppress NF-κB signaling, to reduce the malondialdehyde
content and cell necrosis. In vitro, they could inhibit HR-induced H9c2 cell apoptosis and ROS generation by
activating SIRT1/eNOS pathway and inhabiting NF-κB expression. Emphatically, CAPE-oNO2 presented the
stronger cardioprotection than CAPE both in vivo and in vitro. However, NAM and L-NAME eliminated the
CAPE-oNO2-mediated cardioprotection by restraining SIRT1 and eNOS expression, respectively. It suggested that
CAPE-oNO2 ameliorated MIRI by suppressing the oxidative stress, inflammatory response, fibrosis and necro-
cytosis via the SIRT1/eNOS/NF-κB pathway.
Caffeic acid o-nitro phenethyl ester (CAPE-
oNO2)
Myocardial ischemia/reperfusion injury (MIRI)
Reactive oxygen species (ROS)
SIRT1/eNOS/NF-κB pathway
1. Introduction
peroxidation, altering the structural and functional integrity of cells and
causing cell necrosis and apoptosis [3–5]. For another, ROS is a sti-
Cardiac injury after ischemia/reperfusion (IR) contributes to high
rates of morbidity and mortality in patients, which causes the myo-
cardial cellular structure destruction and the myocardial cell energy
metabolism disorder, following by myocardium tissue infarction [1].
Reactive oxygen species (ROS) generated through a series of interacting
pathways in cardiomyocytes and endothelial cells is greatly increased in
the post-ischemic heart and serves as a critical central mechanism of
post-ischemic injury [2]. When ischemia or myocardial infarction oc-
curs, oxygen free radical formation increases and scavenging system
limits, the scavenging of superoxide anion (O2·−) and hydrogen per-
oxide (H2O2) reduce, transforming oxygen free radical into the stron-
gest oxidation hydroxyl radical (HO·). ROS trigger the chain lipid
mulatory signal of nuclear factor kB (NF-κB) activation which can
trigger inflammation and damage though up-regulating tumor necrosis
factor-α (TNF-α), Bcl-2 associated X Protein (Bax) and transforming
growth factor β1 (TGF-β1) [6]. Oxidative stress and inflammatory re-
sponse play vital roles in the processes of apoptosis and fibrosis, which
are also involved in the pathogenesis of myocardial IR injury (MIRI)
[7–9]. Therefore, the ROS generation and NF-κB activation are the main
factors that determine the severity of myocardial damage.
Silent information regulator 1 (SIRT1), a multifunctional nicotina-
mide adenine dinucleotide (NAD+) dependence protein deacetylase,
acts a pivotal part in the DNA damage repair [10], anti-apoptosis [11],
energy metabolism [12], mitochondrial function protection [13],
⁎
Corresponding author.
E-mail address: lizhubo2004@163.com (Z. Li).
co-first authors.
1
https://doi.org/10.1016/j.redox.2017.11.023
Received 31 October 2017; Received in revised form 16 November 2017; Accepted 27 November 2017
Availableonline29November2017
2213-2317/©2017TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/BY-NC-ND/4.0/).

D. Li et al.
RedoxBiology15(2018)62–73
Fig. 1. Chemical structure of the compounds used
in the present study, CAPE-oNO2 synthetic routes
and IR model establishment methods. (A)
Chemical structure of CAPE, CAPE-pNO2 and CAPE-
oNO2. (B) The synthetic routes of CAPE-oNO2. (C)
The IR rat model was established by 30 min of LAD
ligation and 2 h of reperfusion. (D) H9c2 cells were
subjected to 2 h of hypoxia followed by 4 h of reox-
ygenation to induce MIRI. CAPE, caffeic acid phe-
nethyl ester; CAPE-pNO2, caffeic acid p-nitro phe-
nethyl ester; CAPE-oNO2, caffeic acid o-nitro
phenethyl ester; IR, ischemia/reperfusion; LAD, left
anterior descending coronary artery; MIRI, myo-
cardial ischemia/reperfusion injury.
immunoregulation [14] and resistance to oxidative stress [15] by sti-
mulating the deacetylation of its substrates such as NF-κB, endothelial
nitric oxide synthase (eNOS), peroxisomal proliferators-activated re-
ceptor γ-coactivator-1α (PGC-1α). The deacetylation of NF-κB and PGC-
1α inhibit the NF-κB transcription and improve PGC-1α activity [16],
leading to ROS generation reduction, inflammatory cytokines inhibition
and cell death decreasing. The cardioprotective effect of SIRT1 has been
well demonstrated. Curcumin pretreatment attenuated MIRI by de-
creasing IR-induced mitochondrial oxidative damage through the SIRT1
signaling activation. Similarly, resveratrol (a SIRT1 agonist) treatment
reduced MIRI via SIRT1 activation while Nicotinamide (NAM, a SIRT1
inhibitor) aggravated the myocardial cell apoptosis in dose dependent
manner, indicating that SIRT1 can palliate myocardial cells apoptosis
[17–20].
Caffeic acid phenethyl ester (CAPE, Fig. 1A), a major phenolic ac-
tive ingredient of propolis, has a wide range of pharmacological ac-
tivities such as anti-microbial, anti-tumor, anti-oxidant, anti-in-
flammatory, and so on [21]. Due to its capabilities of scavenging ROS
and immunomodulatory, CAPE has been reported to contribute to
cardioprotection both in vitro and in vivo studies, including attenuation
of cardiomyocyte apoptosis and reduction of myocardial infarction size
[22]. Caffeic acid p-nitro phenethyl ester (CAPE-pNO2, Fig. 1A), a CAPE
analogue with para nitro replace, was determined to be an anti-platelet
agent and a protector of myocardial ischemia with more potent effects
in our previous researches [23,24]. According to the SYBYL molecular
simulation software, nitro is the functional group in the cardioprotec-
tion, in addition, caffeic acid o-nitro phenethyl ester (CAPE-oNO2,
Fig. 1A), synthesized by adding a nitro moiety to the ortho position in
the present study has higher solubility and lower viscera toxicity
(Suppl.Fig. 1). So we selected CAPE-oNO2 to investigate its potential
cardioprotective effect and mechanism on MIRI in this study. As men-
tioned above, curcumin and resveratrol are polyphenol compounds and
stimulate the SIRT1 activity to reduce MIRI. CAPE and CAPE-oNO2, also
possessing polyphenol structure, have yet to be studied with regard as
SIRT1 activation. Therefore, a specific SIRT1 inhibitor nicotinamide
(NAM) had used to elucidate whether CAPE and CAPE-oNO2 can agitate
SIRT1 signaling in this study.
Nitric oxide (NO), a kind of endothelial diastolic factor, is important
for maintaining the constant diastolic state of the cardiovascular
system, regulating the coronary artery base tension and holding the
perfusion of myocardial blood flow [25]. Mollaoglu H found that CAPE
improved the level of NO to exert protective effect on Cd-induced hy-
pertension mediated cardiac impairment in the rats [26]. eNOS, an
enzyme responsible for NO generation, may be of tremendous value for
MIRI. Following NOS inhibition with Nω-nitro-L-arginine methyl ester
(L-NAME), coronary blood flow is prevented while over-expression of
eNOS accelerates functional recovery [27,28]. The results of the pre-
viously study indicated that nitro-substitution CAPE (CAPE-NO2) fur-
ther increased myocardial survival, protected cardiac function fol-
lowing myocardial ischemia and increased NO content as compared
with CAPE [24]. Since the only change of CAPE-NO2 structure is the
introduction of nitro group, we hypothesized the reason of the stronger
cardioprotection of CAPE-NO2 may be the more NO acceleration which
is directly metabolized from CAPE-NO2 or indirectly derived from
eNOS. In order to verify the source of NO and the contribution of eNOS
on MIRI, we have investigated the eNOS expression and NO level by
pretreatment with the eNOS inhibitor L-NAME in the present study.
2. Materials and methods
CAPE was purchased from Meilun Biotechnology (Dalian, China).
CAPE-oNO2 was synthesized firstly in the present study.
Triphenyltetrazolium chloride (TTC), trypsin, penicillin, streptomycin,
dimethyl sulfoxide (DMSO), albumin from bovine serum (BSA), 3-[4, 5-
dimethyl-2-thiazolyl]-2, 5-diphenyl-2-tetrazolium bromide (MTT) were
purchased from Sigma chemicals (St. Louis, MO, USA). The lactate
dehydrogenase (LDH), alpha-hydroxybutyrate dehydrogenase (HBDH),
creatine Kinase (CK), creatine kinase isoenzymes (CK-MB) and ischemia
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D. Li et al.
RedoxBiology15(2018)62–73
modified albumin (IMA) assay kits were purchased from Maccura
Biotechnology (Chengdu, China). The malondialdehyde (MDA), mye-
loperoxidase (MPO), total superoxide dismutase (T-SOD), NO assay kits
and RIPA lysis buffer were purchased from Nanjing Jiancheng
Biotechnology (Nanjing, China). H9c2 cardiomyocytes were obtained
from the Cell Bank at the Chinese Academy of Sciences (Shanghai,
China). Dulbecco's modified Eagle medium (DMEM) and fetal bovine
serum (FBS) were purchased from Gibco/Invitrogen (Carlsbad, CA,
USA). NAM and L-NAME were purchased form Selleck (USA). Acridine
orange (AO) and 2′,7′-dichlorofluorescein-diacetate (DCFH-DA) were
from Beyotime Biotechnology (Shanghai, China). Antibodies against
silent information regulator 1 (SIRT1), endothelial nitric oxide synthase
(eNOS), B-cell lymphoma-2 (Bcl-2), Bcl-2 associated X protein (Bax),
peroxisomal proliferators-activated receptor γ coactivator-1α (PGC-
4) the oNO2 group: IR rats with CAPE-oNO2 (0.1 mg/kg); 5) the NAM +
oNO2 group: IR rats were pretreated with NAM (1 mg/kg) before CAPE-
oNO2 administration (0.1 mg/kg); 6) the NAM + Veh group: IR rats
were pretreated with NAM (1 mg/kg) and vehicle (1 mL/kg); 7) the L-
NAME + oNO2 group: IR rats with L-NAME (1 mg/kg) and CAPE-oNO2
(0.1 mg/kg); 8) the L-NAME + Veh group: IR rats with L-NAME (1 mg/
kg) and vehicle (1 mL/kg).
CAPE and CAPE-oNO2 were dissolved in the vehicle to a final con-
centration of 0.1 mg/mL. CAPE, CAPE-oNO2 and vehicle were tail in-
travenously injected 15 min prior to ischemia, while NAM and L-NAME
were given at 30 min before ischemia (Fig. 1C).
2.3. Quantification of myocardial infarct size
1α), nuclear respiratory factor-2 (Nrf-2), superoxide dismutase
1
Myocardial infarct size was evaluated by TTC (1% in PBS) staining.
The rat hearts were frozen at −20 °C then sliced into transverse 1-mm-
thick section that was incubated in 1% TTC at 37 °C in the dark for
15 min. The area of the infarcted tissues which were stained white or
pale was demarcated and measured digitally using Image-Pro Plus (IPP,
Version 6.0, Media Cybernetics, Inc., Rockville, MD, USA). Infarct size
was calculated as the ratio of infarcted myocardium to the risk region
× 100%.
(SOD1), NF-kappa-B inhibitor (IκB), phosphorylated-IκB (p-IκB), nu-
clear factor kB P65 (P65), phosphorylated-P65 (p-P65), tumor necrosis
factor-α (TNF-α), interleukin-6 (IL-6), transforming growth factor β1
(TGF-β1), mothers against decapentaplegic homolog 2/3 (Smad23),
phosphorylated-Smad23 (p-Smad23), Collagen, GAPDH and horse-
radish peroxidase (HRP)-conjugated goat anti-rabbit secondary anti-
body were purchased from Proteintech Group Inc (Wuhan, China).
2.1. Synthesis and characterization of CAPE-oNO2
2.4. Determination of cardiac marker enzymes in serum
Firstly, a mixure of caffee acid (0.9 g, 5.0 mmol, Ⅰ) in thionyl
chloride (SOCl2, 20 mL) was heated at 80 °C until the reaction fluid
clarified and then distilled under vacuum to remove the excess SOCl2
completely to obtain the intermediate product (caffeoyl chloride, Ⅱ).
Secondly, a solution of caffeoyl chloride in dichloromethane (DCM,
20 mL) was added o-nitrophenylethanol (0.8 g, 5.0 mmol, Ⅲ) which
was dissolved in 20 mL DCM at room temperature. Next, a catalyst
triethylamine TEA, 0.4 mL, 2.9 mmol）was added dropwise, and then
the mixture was stirred at 80 °C for 3 h. Finally, the reaction mixture
was evaporated under vacuum, and the crude product was purified by
silica gel column chromatography (acetone/petroleum ether, 1:4, v/v).
The final products were recrystallized from acetone to obtain pure
crystals (Fig. 1B). 1H (CD3COCD3, 400 MHz) and 13C (CD3COCD3,
100 MHz) nuclear magnetic resonance (NMR) spectra were recorded on
a spectrometer (Ascend 400, Bruker, USA) and liquid chromatography-
mass spectrum (LC-MS) were determined on a mass spectrometer
(LCMS-803010500, Shimadzu, Japan).
Serum samples were isolated from the blood samples by cen-
trifugation (3000 rpm, 4 °C, 10 min). Serum cardiac marker enzymes
including HBDH, LDH, CK, CK-MB and IMA were measured using
spectrophotometric kits by automatic biochemical analyzer (Hitachi,
Japan).
2.5. Determination of SOD, MPO activity and MDA, NO content in tissue
The heart tissues were homogenized (10,000 rpm, 4 °C, 5 min) in
normal saline to obtain 10% homogenates. The homogenates were
centrifuged (3000 rpm, 4 °C, 10 min), and the supernatants were col-
lected for subsequently measurements. SOD, MPO, MDA and NO levels
in the tissues were determined by commercially available kits as the
respective manufacturer's protocols.
2.6. HE, SR and IHC staining of the heart tissues
The heart tissues were immersion-fixed in 4% paraformaldehyde
and embedded in paraffin to prepare 5 µm slices. Deparaffinized and
rehydrated slices were stained with haematoxylin-eosin (HE) and sirius-
red (SR), respectively. Images of these stained sections were examined
using a microscope (Olympus, Tokyo, Japan) and photographed with a
digital camera. For immunohistochemical (IHC) analysis, heart tissue
sections (5 µm) were incubated with TNF-α (1:200 dilution) at 4 °C for
12 h and subsequently incubated with anti-mouse HRP reagent for 1 h
at room temperature. The sections were rinsed in PBS, added the dia-
minobenzidine and counterstained with hematoxylin. The images were
acquired using a light microscopy and camera. The collagen deposition
of SR staining and the area of TNF-α positive staining were measured by
IPP.
2.2. Preparation of myocardial IR model
Sprague-Dawley rats, weighting 200–250 g, were purchased from
the Experimental Animal Centre of Chongqing Medical University
[SCXK (Yu) 2015-001]. The animals were housed in a conditioned en-
vironment (22 1 °C, 12 h light/darkness cycle, free access to food and
water). All the animal protocols conformed to the National Institutes of
Health (NIH) guidelines and were approved by the Ethical Committee
for Animal of Southwest University and all efforts were made to mini-
mize animal pain and suffering throughout the studies.
The myocardial IR rat model was induced by ligation/perfusing left
anterior descending coronary artery (LAD) as described in our previous
study [24]. Under anesthesia, the animals in a tracheotomy had an
electrocardiogram record during the procedure. Then the chest was
opened to expose the heart and the LAD was ligated using a 6-0 silk
suture slipknot. After ischemia for 30 min, the LAD allowed to recover
blood flow for 2 h with slipknot release. At the end of reperfusion,
blood was collected and the heart was quickly removed. Rats were
randomly divided into eight groups, 8 rats in each group: 1) the Con
group, rats were treated with the vehicle (DMSO diluted with normal
saline to a final concentration of 0.01%, 1 mL/kg) and a suture was
passed under the LAD without occlusion; 2) the IR group: IR rats were
treated with vehicle; 3) the CAPE group: IR rats with CAPE (0.1 mg/kg);
2.7. Cell culture and Preparation of cardiomyocytes IR model
For normal cell culture, H9c2 cell was cultured in high glucose
DMEM medium with 10% FBS, penicillin (100 U/mL) and streptomycin
(100 μg/mL) in a humidified incubator (5% CO2, 95% air, 37 °C). Upon
reaching 90% confluency, the cells were passaged with 0.25 mM
trypsin/0.03% EDTA.
To establish cardiomyocytes IR model, H9c2 cell were subjected to
2 h of hypoxia followed by 4 h of reoxygenation. To simulate hypoxia,
cells were cultured in a hypoxic incubator (5% CO2, 95% N2, 37 °C) and
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D. Li et al.
RedoxBiology15(2018)62–73
Fig. 2. Effects of CAPE-oNO2 on reducing infarct size and structural abnormality in the IR rat heart. (A) Results of TTC staining. Red-stained areas represent normal tissue and
unstained pale areas indicate infarcted tissue. Infarct size as a percentage of total volume. Data are expressed as the mean standard deviation (n = 8). *p < 0.05 vs. the control group;
#p < 0.05 vs. the IR group; &p < 0.05 vs. the CAPE group; +p < 0.05 vs. the oNO2 group. (B) Results of HE staining (×200). TTC, triphenyl tetrazolium chloride; HE, haematoxylin-eosin.
the culture medium was replaced with low glucose DMEM medium
without FBS; Reoxygenation was conducted with the normal culture
method. The control group cells kept in the normal incubator without
the hypoxic/reoxygenation (HR) stimulus. CAPE and CAPE-oNO2 were
dissolved in the vehicle (DMSO diluted with culture medium to a final
concentration of 0.01%) and administered at the onset of reoxygena-
tion; NAM and L-NAME were dissolved in the vehicle and given at the
onset of hypoxia (Fig. 1D).
detected by a fluorescence microscope (BD Biosciences, USA). The
fluorescence intensity of ROS was quantified by IPP.
2.10. Western blot analysis
The heart tissues were homogenized in RIPA lysis buffer at 4 °C for
5 min to get 10% homogenates. The homogenates and H9c2 cells were
lysed in RIPA buffer at 4 °C for 45 min, centrifuged (10,000 rpm, 4 °C,
5 min) and collected the supernatant for next experiment. The BCA
protein assay kit was used to measure total protein concentration ac-
cording to the manufacturer's protocol. Protein samples were separated
on SDS-PAGE gels and transferred to PVDF membranes (Bio-Rad, U.S.).
Following the membranes were blocked in tris-buffered saline and
Tween 20 (TBST) containing 5% nonfat dry milk for 1.5 h and then
blotted with SIRT1, GAPDH, eNOS, TGF-β1, P65, p-P65, IκB, p-IκB,
PGC-1α, Nrf1, SOD1, IL-6, TNF-α, Bcl-2, Bax, Smad2/3, p-Smad2/3 and
Collagen (1:2000) primary antibodies overnight at 4 °C. The mem-
branes were washed with TBST and then incubated with the corre-
sponding horseradish peroxidase (HRP)-conjugated secondary anti-
bodies (1:5000) for 1.5 h at room temperature. After the membranes
were washed again, bound antibodies were visualized with enhanced
ECL reagent and quantifed via densitometric analysis using Quantity
One software (Bio-Rad Laboratories, U.S.). Detection of GAPDH was
used as a control.
2.8. Cell toxicity and viability assay
H9c2 cells were seeded into 96-well plates at 1 × 104 cells/well and
incubated for 24 h. To test the cell toxicity of CAPE-oNO2 and vehicle,
cells were given CAPE-oNO2 or vehicle and cultured for 4, 8, 24 h, re-
spectively. To evaluate the cell viability of CAPE and CAPE-oNO2, cells
were subjected to HR and given CAPE and CAPE-oNO2. Next, the
number of viabile cell was evaluated by MTT assay. Briefly, the cells
were incubated with MTT at a final concentration of 0.5 mg/mL at the
end of reoxygenation. After 4 h, the number of viable cells was mea-
sured by evaluating absorbance at 490 nm with a microplate reader
(Tecan Infnite M1000, Austria).
2.9. AO and ROS staining
H9c2 cells were seeded into 6-well plates at 1 × 106 cells/well and
incubated for 24 h followed by HR. For evaluation of apoptosis, cells
were stained with AO dye solution (100 μg/mL) after reoxygenation.
For measurement of ROS generation, cells were incubated with DCFH-
DA (10 μmol/L) at 37 °C for 20 min. The apoptosis cells and ROS were
2.11. Statistical analysis
Data were analyzed using Statistic Package for Social Science (SPSS,
version 16.0) statistical analysis software. Data One-way analysis of
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D. Li et al.
RedoxBiology15(2018)62–73
variance (ANOVA) with the Student–Newman–Keuls post-test was used
for comparisons of data between groups. P-values below 0.05
(p < 0.05) were considered indicative of statistical significance.
3. Results
3.1. Structural characterization of CAPE-oNO2
CAPE-oNO2: light brown solid, melting point 148–152 °C. 1H NMR
(CD3COCD3, 400 MHz): δ 3.33(2 H, m, CH2), 4.43(2 H, t, J = 6.48,
CH2), 6.23(1 H, d, J = 15.96, α-H), 7.50(1 H, d, J = 15.96, β-H),
6.87(1 H, d, J = 8.19, Ph-H), 7.03(1 H, dd, J = 8.09, 2.20, Ph-H),
7.15(1 H, dd, J = 4.15, 2.09, Ph-H), 7.52(1 H, d, J = 1.53, Ph-H),
7.61(1 H, d, J = 7.73, 1.53, Ph-H), 7.68(1 H, td, J = 7.51, 1.37, Ph-H),
7.96(1 H, dd, J = 8.16, 1.32, Ph-H). 13C NMR (CD3COCD3, 400 MHz):
δ 31.7, 63.4, 114.3, 114.4, 114.5, 115.5, 121.7, 124.5, 126.7, 128.0,
132.8, 132.9, 133.0, 145.0, 145.4, 147.9, 166.2. LC-MS m/z (%):
328.0724[M-H]+ (calc. C17H15NO6: 329.02) (Suppl. Fig. 1).
3.2. CAPE and CAPE-oNO2 protected against myocardial injury in the IR
rats
The infarct size of heart extended after the IR procedure, CAPE and
CAPE-oNO2 caused a significant lessening in the infarct size, and the
lessening degree of CAPE-oNO2 was more than that of CAPE (p < 0.05).
However, pretreated with L-NAME and NAM could effectively abolish
the alleviative effect of CAPE-oNO2 on the infarct size (p < 0.05,
Fig. 2A). Furthermore, HE staining shows that the cardiac tissue ex-
hibited a clear and well organized structure little inflammatory in-
filtration or cardiac necrosis in the control, but myocardial structural
abnormalities histological changes including perinuclear vacuolization,
necrosis and massive inflammatory infiltration were detected in the IR
group, the myocardial structural abnormalities were almost un-
detectable in the CAPE and the CAPE-oNO2 group. In contrast, pre-
treated with NAM and L-NAME could neutralize the ameliorating effect
of CAPE-oNO2 (Fig. 2B).
The measurement of cardiac enzymes as outlined in Table 1, the
activities of LDH, HDBH, CK, CK-MB and MIA were significantly in-
creased in the IR group (p < 0.05). Treating with CAPE-oNO2 decreased
cardiac enzymes release much stronger than CAPE did (p < 0.05).
Conversely, there were no marked differences among the L-NAME,
NAM group and the IR group.
3.3. CAPE and CAPE-oNO2 motivated SIRT1 and eNOS expression in the
IR rats
As shown in Fig. 3, the expression of SIRT1, eNOS and Bcl-2, and NO
level were markedly decreased (p < 0.05). CAPE and CAPE-oNO2 in-
creased significantly the content of SIRT1, eNOS and NO (p < 0.05),
and the up-regulation of CAPE-oNO2 on SIRT, eNOS and NO was higher
than those of CAPE (p < 0.05). Inversely, pretreated with NAM and L-
NAME eliminated the CAPE-oNO2-mediated rise of SIRT1, eNOS, Bcl-2
and NO level, respectively (p < 0.01). The Bax expression was the op-
posite.
3.4. CAPE and CAPE-oNO2 attenuated cardiac oxidative stress in the IR rat
After IR operation, the IR group had a significantly increasing in
MDA content (Fig. 4A) and a significantly decreasing in T-SOD activity
((Fig. 4B, p < 0.05), and CAPE and CAPE-oNO2 administration mark-
edly decreased these enzyme activity (p < 0.05). However, neither
MDA content nor SOD activity had significant changes in the NAM and
L-NAME group compared with the IR group. Western blot analysis
showed that the expression of antioxidant responsive protein which
including PGC-1α, Nrf-2 and SOD1 were decreased markedly after IR
produce (p < 0.05) (Fig. 4C). Treatment with CAPE and CAPE-oNO2
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RedoxBiology15(2018)62–73
Fig. 3. Effects of CAPE-oNO2 on SIRT1, eNOS, Bcl-2 and Bax expression, and NO level of the IR rat heart. (A) Representative images of the Western blots are shown. GAPDH was
used as an internal control. (B) The NO level in the heart tissue was determined by commercially available kits. Data are expressed as the mean standard deviation (n = 8). *p < 0.05
vs. the control group; #p < 0.05 vs. the IR group; &p < 0.05 vs. the CAPE group; +p < 0.05 vs. the oNO2 group. SIRT1, silent information regulator 1; eNOS, endothelial nitric oxide
synthase; Bcl-2, B-cell lymphoma-2; Bax, Bcl-2 Associated X Protein.
activated PGC-1α, Nrf-2 and SOD1. However, CAPE-oNO2 didn’t in-
crease the PGC-1α, Nrf-2 and SOD1 expression in the presence of NAM
or L-NAME.
group (p < 0.05). Treatment with CAPE and CAPE-oNO2 decreased
these proteins expression, and the reduced degree of CAPE-oNO2 was
much heavier than that of CAPE (p < 0.05). However, the down-reg-
ulation effects of these proteins didn’t appear in the NAM and the L-
NAME group (Fig. 5C).
3.5. CAPE and CAPE-oNO2 remitted cardiac inflammation in the IR rat
In IHC staining (Fig. 5A), TNF-α level was meaningfully raised in
the IR group (p < 0.05), and CAPE and CAPE-oNO2 settlement abated
tissue TNF-α level (p < 0.05). L-NAME and NAM disposing elevated
TNF-α level compared with CAPE-oNO2 group (p < 0.05). MPO activity
was markedly fortified after the IR, treatment with CAPE and CAPE-
oNO2 diminished tissue MPO levels (p < 0.05), whereas the MPO con-
tent wasn’t decreased by CAPE-oNO2 in the presence of L-NAME and
NAM (Fig. 5B, p < 0.05). Moreover, western blot analysis indicated that
p-P65/P65, p-IκB/IκB, IL6 and TNF-α dramatically lifted in the IR
3.6. CAPE and CAPE-oNO2 attenuated cardiac fibrosis in the IR rat
In SR staining (Fig. 6A), significant collagen deposition in heart
tissue was seen in IR group, and the collagen deposition could be ob-
viously weakened by CAPE and CAPE-oNO2 therapy (p < 0.05). How-
ever, CAPE-oNO2 had little effects on inhibiting cardiac collagen de-
position with NAM and L-NAME pretreatment.
Meanwhile, the western blot showed that TGF-β1, p-Smad23/
Smad23 and Collagen expression increased after the IR injury
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Fig. 4. Effects of CAPE-oNO2 on attenuating cardiac oxidative stress in the IR rat. (A) Production of MDA in heart tissue. (B) The activity of T-SOD in heart tissue. (C) The expression
of PGC-1α, Nrf-2 and SOD1 were examined by Western blot assay and quantified by densitometric analysis. GAPDH was used as an internal control. Data are expressed as the
mean standard deviation (n = 8). *p < 0.05 vs. the control group; #p < 0.05 vs. the IR group; &p < 0.05 vs. the CAPE group; +p < 0.05 vs. the oNO2 group. MDA, malondialdehyde; T-
SOD, total superoxide dismutase; PGC-1α, peroxisomal proliferators-activated receptor γ coactivator-1α; Nrf-2, nuclear respiratory factor-2; SOD1, superoxide dismutase 1.
(p < 0.05). In the CAPE and CAPE-oNO2 group displayed a significant
reduction of TGF-β1, p-Smad23/Smad23 and Collagen expression
(p < 0.05). However, the expression of TGF-β1, p-Smad23/Smad23 and
Collagen weren’t lessened in the NAM and L-NAME group (vs the CAPE-
oNO2 group, p < 0.05, Fig. 6B).
SOD1 and Bcl-2 protein level markedly decreased in the HR group
(p < 0.05). CAPE and CAPE-oNO2 treatment increased the three pro-
teins expression with dose-dependent. However pretreatment with
NAM and L-NAME eliminated the rise of those proteins level, respec-
tively (p < 0.05). The trend of p-P65/P65, p-IκB/IκB and Bax were the
opposite.
3.7. CAPE and CAPE-oNO2 prevent HR injury in H9c2 cell model
4. Discussion
MTT assay showed that there was no significant difference in cell
viability between CAPE-oNO2 (0, 1, 10, 20, 40, 60, 80, 100 μM) groups
and the control in spite of cultivation for 4, 8 and 24 h (Fig. 7A). After
HR, a remarkable debasement in cell viability compared to control
group were observed in MTT assay. Treatment with CAPE and CAPE-
oNO2 at concentrations of 10–40 μM increased cell viability in a dose-
dependent manner. However, the cell viability gradually decreased
after treating with CAPE and CAPE-oNO2 at concentrations of 60 and
80 μM, compared with 40 μM CAPE and CAPE-oNO2 administration,
respectively. Besides, the cell viability of CAPE group was lower than
that of CAPE-oNO2 group (p < 0.05, Fig. 7B).
And then, as shown in AO staining (Fig. 7C), a large number of
normal H9c2 cell were stained green and the nucleus were clear and
intact in the control, the number of viable cells decreased significantly
and the characteristics of cell apoptosis, including chromosome fixa-
tion, fragmentation, and sparse cytoplasm were detected in the HR
group. Treatment with CAPE and CAPE-oNO2 increased the viable cell
quantity and remitted HR-induced cardiomyocytes apoptosis. However,
the protection effect of CAPE-oNO2 was each partly suppressed by L-
NAME and NAM. As ROS staining (Fig. 7D) shown that HR-induced
ROS rise was prominently restrained by CAPE or CAPE-oNO2 treatment
in a dose-dependent manner. But pretreatment with NAM and L-NAME,
CAPE-oNO2 couldn’t excellently cut down the ROS production.
MIRI results in the cardiac contractile dysfunction, apoptotic and
necrotic cell death, leading to fibrosis and irreversible myocyte damage
[29]. The mechanism of MIRI is complex and involves that reperfusion
exacerbates cell ROS generation, oxidative stress increase and various
downstream transcription factors activations, including NF-κB, TNF-α
and TGF-β1, to accelerate the pro-inflammatory and cell death path-
ways [30]. Therefore, suppression of oxidant stress and inflammation
pathways holds great promise for alleviating cardiac fibrosis and cell
death. Correspondingly, increasing evidence indicates that CAPE is an
effective anti-inflammatory and antioxidant, and the two main prop-
erties of CAPE make it a promising cardioprotective efficacy. The pro-
posed mechanism is seen as an attenuation of cardiomyocyte apoptosis
via CAPE-inhibition of lipid peroxidation, NF-κB activation and Nrf2
inactivation, and ROS scavenging of CAPE is also involved in pre-
venting the IR-induced apoptotic [31–33]. CAPE-oNO2, a new deriva-
tive of CAPE, was synthesized firstly and characterized in this study,
and it might possess a variety of biological properties and potential
cardiovascular protection on the basis of SYBYL analysis.
In this study, we have evaluated the effects of CAPE-oNO2 on the rat
model of LAD ligation/reperfusion and the cell model of H9c2 cell
cultured in the oxygen-glucose deprivation/reoxygenation (OGD/R)
based on the positive control of CAPE. The results showed that CAPE-
oNO2 treatment conferred a cardioprotective effect on the model rat
and the model cell, as evidenced by decreased myocardial infarct size,
ameliorated the structural abnormalities, reduced serum cardiac
marker enzymes activities, increased oxidation resistance, decreased
the inflammatory response, attenuated collagen deposition and
3.8. CAPE and CAPE-oNO2 agitated SIRT1 and eNOS signaling in the HR
H9c2 cell
Western blot analysis (Fig. 8) showed that SIRT1, eNOS, PGC-1α,
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Fig. 5. Effects of CAPE-oNO2 on remitting cardiac inflammation in the IR rat. (A) Results of IHC staining (×200). The integrated option density value was used to measure the
expression level of TNF-α. (B) The activity of MPO in heart tissue. (C) The expression of IκB, p-IκB, P65, p-P65, TNF-α and IL-6 were examined by Western blot assay and quantified by
densitometric analysis. GAPDH was used as an internal control. Data are expressed as the mean standard deviation (n = 8). *p < 0.05 vs. the control group; #p < 0.05 vs. the IR group;
&p < 0.05 vs. the CAPE group; +p < 0.05 vs. the oNO2 group. IHC, immunohistochemistry; MPO, myeloperoxidase; TNF-α, tumor necrosis factor-α; IκB, NF-kappa-B inhibitor; p-IκB,
phosphorylated-IκB; P65, nuclear factor kB P65, phosphorylated-P65 (p-P65), and IL-6, interleukin-6.
improved cell viability. More importantly, CAPE-oNO2 exerted the
much more capacity both in vivo and in vitro.
IκB-α and inhibiting NF-κB DNA binding through S-nitrosylation of
cysteine residue of the p50 subunit [39]. There is report that the re-
sveratrol-mediated activation of SIRT1 increased MnSOD levels in
cardiomyocytes and suppressed fibrosis, preserved cardiac function,
and significantly improved the survival in TO-2 hamsters [40]. Like-
wise, our results illustrated that the CAPE-oNO2-mediated activation of
SIRT1 increased anti-oxidative effect through PGC-1α system sensiti-
zation, decreased inflammatory response through NF-κB inactivation,
suppressed fibrosis through TGF-β1 down-regulation in the IR rats.
Oxidative stress resulted from the IR-induced massive ROS genera-
tion is an important pathogenesis of MIRI [41], restraining the propa-
gation of the oxidative chain reaction and direct decreasing the free
radical levels could be significant to prevent IR-induced myocardial
injury. The present study showed that IR could aggravate myocardial
oxidative stress. CAPE-oNO2 significantly diminished the MDA level,
intensified the T-SOD activity and increased the proteins expression of
PGC-1α, Nrf1, SOD1 in the IR rat hearts. It implied that CAPE-oNO2 as a
CAPE homolog also possess antioxidant activity that was involved in
the cardio-protection on MIRI. MDA is an end-product of lipid perox-
idation while SOD is an enzyme for scavenging ROS, they all could
reflect the lipid peroxidation degree. Antioxidant responsive gene SOD1
is induced by the Nrf system sensitization of PGC-1α and the PGC-1α
expression is incited by SIRT1 and inhibited by NF-κB [42].
In vivo, the results of TTC staining, HE staining and cardiac enzymes
measuring showed that CAPE-oNO2 was able to abolish the raise in
infarct size, the structural abnormalities and the elevation of cardiac
enzymes (CK, CK-MB, LDH, HBDH and MIA), increase the anti-apop-
totic factor Bcl2 expression and reduce the pro-apoptotic factor Bax
expression in the IR rats. These results indicated that CAPE-oNO2 in-
creased myocardial survival and protected cardiac structure following
myocardial ischemia, which was basically consistent with CAPE-pNO2
based on Duqin's research [24]. Moreover, CAPE-oNO2 could stimulate
the activities of SIRT1 and eNOS more excellently than CAPE could do,
resulting in the stronger cardioprotection of CAPE-oNO2. SIRT1 has an
unequivocal protective demonstrated both in animal experiments and
human studies [34]. The PGC-1α agitation of SIRT1 may protect against
ischemia-induced oxidative damage [35], the level of P65 acetylation
and TGF-β1 expression are inhibited by SIRT1 [36], and the phos-
phorylation of eNOS was significantly higher in an isolated myocardial
IR model of SIRT1-overexpressing mice than in lack of SIRT1 mice [37].
Furthermore, eNOS that enhance NO release prior to ischemia protects
against MIRI by preserving ischemic blood flow, attenuating platelet
aggregation and leukocyte adherence [38]. Besides, NO has been re-
ported to suppress NF-κB activation by stabilizing the NF-κB inhibitor
69

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Fig. 6. Effects of CAPE-oNO2 on attenuating cardiac fibrosis in the IR rat. (A) Collagen deposition was measured by SR staining (×200), followed by semi-quantitative analysis. (B)
The expression of TGF-β, Smad23, p-Smad23 and collagen were examined by Western blot assay and quantified by densitometric analysis. GAPDH was used as an internal control. Data
are expressed as the mean standard deviation (n = 8). *p < 0.05 vs. the control group; #p < 0.05 vs. the IR group; &p < 0.05 vs. the CAPE group; +p < 0.05 vs. the oNO2 group. SR,
Sirius-red; TGF-β1, transforming growth factor β1; Smad23, mothers against decapentaplegic homolog 2/3; p-Smad23, phosphorylated-Smad23.
CAPE-oNO2 could significantly rebate MPO and the major in-
flammatory gene expression including p-IκB/IκB, p-P65/P65, IL6 and
TNF-α in the IR rats heart. ROS stimulates NF-κB activation by pro-
moting IκB phosphorylation and degradation, which is one of the main
ways of regulating cardiac inflammation and damage [43]. The dis-
assembly of IκB and NF-κB allow -P65 subunit to translocate into nu-
clear and subsequently motivate the specific promoter sequence of the
target genes, such as TNF-α, IL6, Bcl-2 and Bax, triggering in-
flammatory injury and apotosis [44]. Our results demonstrated that
CAPE-oNO2 possessed the anti-inflammatory effect through inhabiting
IκB/P65 pathway activation.
CAPE-oNO2 could remit collagen deposition through suppressing
TGF-β1, p-Smad23/ Smad23 and Collagen. TGF-β1 whose expression is
triggered by P65 and limited by SIRT1, makes myocardial fibroblasts to
secrete copious collagen depositing in myocardial interstitium, causing
the imbalance of collagen and fibrosis [45–47]. Moreover, ROS not only
induce cardiomyocyte hypertrophy, apoptosis through TGF-β1 activa-
tion but also lead to vascular endothelial dysfunction through NO ex-
haustion, which aggravate the occurrence of myocardial fibers [48]. It
indicated that the TGF-β1/Smad pathway was directly inhibited by the
CAPE-oNO2-mediated SIRT1 activation and ROS scavenging, and that
the P65 inactivation and eNOS activation of SIRT1 could also palliate
the cardio fibrosis.
In vitro, The MTT assay testified that CAPE-oNO2 and vehicle had no
cell toxicity at concentrations from 1 to 100 μM regardless of culturing
for 4, 8 and 24 h. After HR, CAPE-oNO2 was able to increase cell vi-
abilities in a dose-dependent manner at concentrations from 10 to
40 μM. However, the cell viabilities were gradually decreased at con-
centrations from 60 and 80 μM, it may because that when the con-
centration of CAPE-oNO2 overly increased, excessive NO was produced
by eNOS overexpression to aggravate damage [36]. Therefore we used
the concentrations from 10 to 40 μM for the next research. The results
of AO staining and DCFH-DA staining showed that CAPE-oNO2 could
reduce the HR-induced apoptosis and ROS generation, respectively.
West-blot analysis exhibited the SIRT1, eNOS, Bcl-2, SOD1 up-regula-
tions and p-IκB/IκB, p-P65/P65 down-regulations. These results de-
noted that CAPE-oNO2 increased survival, reduced apoptosis and ROS
on HR H9c2 cell through SIRT1 agitation.
To further confirm that the SIRT1 and eNOS pathway play a critical
role on the cardio-protective effect of CAPE-oNO2, we have pretreated
with SIRT1 inhibitor NAM and the NOS inhibitor L-NAME before CAPE-
oNO2 or vehicle administration, respectively. As anticipation, SIRT1
and eNOS expression were suppressed and the CAPE-oNO2-mediated
cardioprotection was largely eliminated in the presence of the NAM and
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Fig. 7. Effects of CAPE-oNO2 on inhibiting cell apoptosis and ROS generation cell in the HR H9c2 cardiomyocytes. (A) To investigate the cytotoxicity of CAPE-oNO2, H9c2
cardiomyocytes were treated with 0, 5, 10, 20, 40, 60, 80 and 100 μM CAPE-oNO2 for 4, 8 and 24 h, followed by MTT analysis. After 2 h of hypoxia, H9c2 cell were treated with different
concentrations of CAPE and CAPE-oNO2 in the absence or presence of NAM and L-NAME, followed by 4 h of reoxygenation. (B) The cell viability was measured with MTT assays. (C) The
cell apotosis was detected with AO staining. (D) The ROS generation was stained with DCFH-DA. Data are expressed as the mean standard deviation (n = 8). *p < 0.05 vs. the control
group; #p < 0.05 vs. the IR group; &p < 0.05 vs. the CAPE group; +p < 0.05 vs. the oNO2 group. ROS, reactive oxygen species; NAM, nicotinamide; L-NAME, Nω-nitro-L-arginine methyl
ester, MTT, 3-[4, 5-dimethyl-2-thiazolyl]−2, 5-diphenyl-2-tetrazolium bromide; DCFH-DA, 2′,7′-dichlorofluorescein-diacetate.
L-NAME both in vivo and in vitro. Interestingly, SIRT1 expression is
only stifled by NAM while eNOS expression is suppressed by both two
inhibitors, along with the alternation of P65, PGC-1α, TGF-
β1downstream pathway, which manifested that SIRT1 may be an up-
stream of eNOS, and both SIRT1 and eNOS could down-regulate P65
signaling. After pretreatment of NAM and L-NAME, the CAPE-NO2-
mediated NO increase was also eliminated, which indicated that NO
may be derived from eNOS rather than directly metabolized from
CAPE-NO2. The previous studies in our lab also showed that CAPE-NO2
couldn’t metabolize out NO. Lai XY used a mixture system of rat liver
microsome and CAPE-NO2 to build a hepatic metabolism model in vitro
and speculate that the main metabolic products of CAPE-NO2 included
caffeic acid, ferulic acid, nitro phenylethyl alcohol and ferulic acid nitro
phenethyl ester [49]. CAPE-NO2 was transformed into C14H16N2O6,
C8H11O4P, C10H14N2O6S or C16H11NO7 rather than denitration meta-
bolites in the HT-29 cells Xenografts athymic mice heart, liver, spleen
and kidney [50]. What's more, Yao XF revealed that the major meta-
bolic pathway of CAPE-NO2 may be a phase II reaction (biotransformed
by glucuronidation, sulfonation or methylation), although a small
quantity of them could be metabolized via a phase I reaction (hydro-
lysis) [51].
Summary, SIRT1 stimulates the deacetylation of NF-κB, PGC-1α and
eNOS, leading to the inhibition of NF-κB transcription and the promo-
tion of PGC-1α and eNOS activities. NO derived by eNOS suppresses
NF-κB activation as well. Afterwards, the NF-κB inhibition, results in
that oxidative stress reduces through PGC-1α/Nrf1 system sensitiza-
tion, inflammatory response abates through TNF-α and IL-6 up-reg-
ulation, collagen deposition diminishes through TGF-β1/Samd signal
inhibition, and necrocytosis lessens through Bcl-2 intensifying and Bax
depressing. It signified that CAPE-oNO2 exert cardioprotective effect on
anti-oxidatant, anti-inflammatory, collagen deposition and anti-apop-
tosis via the SIRT1/ eNOS/ NF-κB signaling agitation. The stronger
cardioprotection of CAPE-oNO2 may attribute to the more powerful
agitation of SIRT1/ eNOS/ NF-κB signaling (Fig. 9).
71

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Fig. 8. Effects of CAPE-oNO2 on SIRT1/eNOS pathway proteins expression in the HR H9c2 cells. The expression of SIRT1, eNOS，PGC-1α，p-IκB, IκB, p-P65, P65,SOD1, Bcl-2 and
Bax were examined by Western blot assay and quantified by densitometric analysis. GAPDH was used as an internal control. Data are expressed as the mean standard deviation (n = 8).
*p < 0.05 vs. the control group; #p < 0.05 vs. the IR group; &p < 0.05 vs. the CAPE group; +p < 0.05 vs. the oNO2 group.
against MIRI in the absence of NAM and L-NAME. In vivo, CAPE-oNO2
ameliorated MIRI by suppressing the oxidative stress, inflammatory
response, fibrosis and necrocytosis via the SIRT1/eNOS/NF-κB/
pathway. In vitro, CAPE-oNO2 inhibits HR-induced cell apoptosis and
ROS production by activating SIRT1/eNOS/NF-κB. Emphatically,
CAPE-oNO2 presented a stronger cardioprotective effect than CAPE
both in vivo and in vitro while NAM and L-NAME eliminates the CAPE-
oNO2-mediated cardioprotection. These results reveal that CAPE-oNO2
may have tremendous therapeutic potential in the treatment of MIRI.
Acknowledgments
We would like to thank the Shanxi Zhaoyi Biological Co. Ltd., China
(SWU2013130) and the Fundamental Research Funds for the Central
Universities (XDJK2017A008) for financial support.
Disclosures
The authors have no disclosures to declare.
Author contributions
Fig. 9. Cardioprotective effect of CAPE-oNO2. CAPE-oNO2 ameliorated MIRI by sup-
pressing the oxidative stress, inflammatory response, fibrosis and necrocytosis via the
SIRT1/eNOS/NF-κB pathway. IR, ischemia/reperfusion; HR, hypoxia/reoxygenation; L-
NAME, eNOS inhibitor Nω-nitro-L-arginine methyl ester; NAM, SIRT1 inhibitor nicoti-
namide; CAPE, caffeic acid phenethyl ester; oNO2, caffeic acid o-nitro phenethyl ester.
Li Z and Li D designed the project; Li D, Wang X and Li S performed
the rat experiments; Li D, Zhou Y and Huang Q took charge the cell
experiments; Li D and Li Z wrote the main manuscripts; H. T. Wang X
analyzed and interpreted data; all authors reviewed the manuscript.
5. Conclusion
Appendix A. Supporting information
Collectively, our findings using SD rat hearts and H9c2 cardio-
Supplementary data associated with this article can be found in the
myocytes suggest that CAPE-oNO2 exerts a profound cardioprotection
online version at http://dx.doi.org/10.1016/j.redox.2017.11.023.
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RedoxBiology15(2018)62–73
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