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Research Article
The Cardioprotective Effects of Citric Acid and L-Malic Acid on
Myocardial Ischemia/Reperfusion Injury
Xilan Tang,1,2,3 Jianxun Liu,1Wei Dong,2,3 Peng Li,1Lei Li,1Chengren Lin,1
Yongqiu Zheng,1Jincai Hou,1and Dan Li1
1Experimental Research Center, Xiyuan Hospital, China Academy of Chinese Medical Sciences, Beijing 100091, China
2Key Laboratory of Modern Preparation of TCM, Jiangxi University of Traditional Chinese Medicine, Nanchang 330004, China
3Beijing University of Chinese Medicine, Beijing 100029, China
Correspondence should be addressed to Jianxun Liu; liujx@sina.com
Received December ; Revised April ; Accepted April
Academic Editor: Hao Xu
Copyright © Xilan Tang et al. is is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Organic acids in Chinese herbs, the long-neglected components, have been reported to possess antioxidant, anti-inammatory, and
antiplatelet aggregation activities; thus they may have potentially protective eect on ischemic heart disease. erefore, this study
aims to investigate the protective eects of two organic acids, that is, citric acid and L-malic acid, which are the main components
of Fructus Choerospondiatis, on myocardial ischemia/reperfusion injury and the underlying mechanisms. In in vivo rat model of
myocardial ischemia/reperfusion injur y, we found that treatments with citric acid and L-malic acid signicantly reduced myocardial
infarct size, serum levels of TNF-𝛼, and platelet aggregation. In vitro experiments revealed that both citric acid and L-malic acid
signicantly reduced LDH release, decreased apoptotic rate, downregulated the expression of cleaved caspase-, and upregulated the
expression of phosphorylated Akt in primary neonatal rat cardiomyocytes subjected to hypoxia/reoxygenation injury. ese results
suggest that both citric acid and L-malic acid have protective eects on myocardial ischemia/reperfusion injury; the underlying
mechanism may be related to their anti-inammatory, antiplatelet aggregation and direct cardiomyocyte protective eects. ese
results also demonstrate that organic acids, besides avonoids, may also be the major active ingredient of Fructus Choerospondiatis
responsible for its cardioprotective eects and should be attached great importance in the therapy of ischemic heart disease.
1. Introduction
Ischemic heart disease is a leading cause of mortality of the
clinical cardiovascular diseases and remains a major public
health threat worldwide. Myocardial damage in ischemic
heart disease is likely due to ischemia/reperfusion injury.
Myocardial ischemia/reperfusion can lead to cardiomyocyte
loss by several pathological mechanisms, which contain free
radical formation, inammatory response and endothelial
dysfunction, platelet aggregation and microembolization,
necrosis and apoptosis, and so forth []. erefore, a phar-
macologic approach to ischemia/reperfusion injury remains
a longstanding challenge in medicine.
Fructus Choerospondiatis,awidelyknownMongolian
herb derived from the dried mature fruit of Choerospondias
axillaris (Roxb.) Burtt et Hill, with ecacy of “activating
vital energy and blood circulation” and “nourishing heart
for tranquilization” [], according to traditional Chinese
medicine theory, has been used extensively as a remedy for
ischemic heart disease and achieved good clinical ecacy.
It consists of several ingredients, including organic acids,
phenolic acids, and avonoids. Previous studies have been
focused on avonoids, which were always considered to be
themainactiveconstituentsresponsibleforthepharma-
cological actions of Fructus Choerospondiatis []. However,
recent pharmaceutical chemistry studies showed that the
content of total avonoids (mainly quercetin) in Fructus Cho-
erospondiatis was very low and only accounted for .%
of its water-soluble extracts [], whereas the content of total
organic acids was in signicant amounts, up to .%. And
citric acid and L-malic acid are two main organic acids of
Fructus Choerospondiatis, the content of which accounted
for .% and .% of total organic acids, respectively
[].
Evidence-Based Complementary and Alternative Medicine
Organic acids, which are also widely distributed in fresh
fruits, vegetables, and spices besides Chinese herbs, were
always considered to be weak in activity and were usually
discarded in the extraction process. erefore, they have been
long neglected and their pharmacological actions have not
been suciently studied. Recent research indicated that some
organicacidshavevariouspharmacologicaleects,including
anti-inammatory response [,], antiplatelet aggregation
[], antioxidant [,], and reducing cell apoptosis and so
on. erefore, we hypothesized that organic acids might have
protective eect on myocardial ischemia/reperfusion injury.
In the present study, we investigated the protective eects
of citric acid and L-malic acid on myocardial ischemia/
reperfusion and its possible mechanisms. To the best of our
knowledge, the nding that citric acid and L-malic acid
have protective eects on myocardial ischemia/reperfusion
injury by anti-inammatory, antiplatelet aggregation, and
direct cardiomyocyte protective eects is therefore partic-
ularly signicant for providing important insights into the
understanding of cardioprotective eects of organic acids in
traditional Chinese medicine.
2. Materials and Methods
2.1. Animals. Male adult Wistar rats (– g body weight)
were provided by the Experimental Animal Research Insti-
tute, Chinese Academy of Medical Sciences (clean degree,
certicate no. SCXK (Jing) -). Neonatal Sprague-
Dawley rats (SPF degree, to days old) were purchased
from Beijing Vital River Laboratory Animal Technology
Co.Ltd., China (certicate no. SCXK (Jing) -). Rats
were housed under standard conditions and supplied with
drinking water and food ad libitum. All animal experiments
in this study were performed in accordance with China
Academy of Chinese Medical Sciences Guide for Laboratory
AnimalsthatconformstotheGuidefortheCareandUseof
Laboratory Animals published by the U.S. National Institutes
of Health (NIH Publications no. -, revised ).
2.2. Reagents and Chemicals. Lactate dehydrogenase (LDH)
assay kit was purchased from Beijing Zhongsheng Biological
Technology Co., Ltd. (batch no. ). Rat tumor necro-
sis factor-𝛼(TNF-𝛼) Quantikine ELISA kit was obtained
from R&D (Catalog no. RTA, USA). FITC-annexin
V/propidium iodide apoptosis detection kit was from BD
Biosciences (Catalog no. , USA). Nitroblue tetrazolium
(N-BT) (Ultra Pure Grade, ) and anti-cleaved caspase-
antibody(Catalogno.C)wereproductsofSigma
Chemical Co. (USA). e antibodies for anti-p-Akt (Ser,
no. ) and anti-Akt (no. ) were from Cell Signaling
(USA). Citric acid (batch no. -) and L-malic
acid (batch no. -) were purchased from National
Institutes for Food and Drug Control (Beijing, China). All
chemicals used were of analytical grade.
2.3. Drug Pretreatment and Myocardial Ischemia/Reperfusion
Protocols. e studies of citric acid and L-malic acid on
myocardial ischemia/reperfusion were performed indepen-
dently although the experimental designs of them were
identical. All animals were randomly assigned to six groups
(𝑛=10for each group). Vehicle or drugs were fed once daily
( mL/kg) for consecutive days prior to the experiment and
treated as follows.
Group : sham group. Rats were orally administered
.% saline.
Group : ischemia/reperfusion (I/R) model control
group. Rats were also orally administered .% saline.
Group : diltiazem (Tanabe Seiyaku Co., Ltd., Tian-
jin)-pretreated group as positive control. Rats were
orally administered diltiazem at a dose of mg/kg.
Group : clopidogrel (Sano Winthrop Industrie)-
pretreated group as another positive control. Rats
were orally administered clopidogrel at a dose of
. mg/kg.
Group : citric acid- or L-malic acid-pretreated group.
Rats were orally administered citric acid or L-malic
acid at a dose of mg/kg.
Group : citric acid- or L-malic acid-pretreated group.
Rats were orally administered citric acid or L-malic
acid at a dose of mg/kg.
Myocardial ischemia/reperfusion injury rat model was
constructed by LAD ligation for min followed by h
reperfusion at h aer the last drug treatment as previously
described []. Rats were anesthetized with .% chloral
hydrate (Sinopharm Chemical Reagent Co., Ltd., China)
( mg/kg, i.p.). e neck was opened with a ventral midline
incision. e trachea was exposed and cannulated to establish
articial respiration provided by a rodent ventilator (ALC-
VS, China) with oxygen at a breath ratio of : and at a
frequency of breaths/min with tidal volume of .mL.
Myocardial ischemia was produced by exteriorizing the heart
through a le thoracic incision and placing a - silk suture
and making a plastic tubing at the distal / of the le anterior
descending coronary artery. Aer min of ischemia, the
plastic tubing was cut and the myocardium was reperfused
for h.
2.4. Measurement of Myocardial Infarct Size. Myocardial
infarct size was evaluated by N-BT staining as previously
described []. Briey, at the end of h reperfusion, rats were
anesthetized with .% chloral hydrate and then sacriced.
e hearts were quickly excised and sliced into sections
from the position under the ligation line. e slices were
weighed and then immediately incubated in N-BT staining
solution dissolved in saline at ∘C for min. e infracted
size, noninfracted size, and total heart size were measured
by multimedia color pathological image analytical system
(MPIAS-, Beijing). e infarction percentage of the ven-
tricle, infarction percentage of the heart, infarction area, and
infarction weight were calculated.
2.5. Determination of Inammatory Cytokine Activity and
Platelet Maximum Aggregation Rate. e serum levels of
TNF-𝛼were measured using ELISA kits according to the
manufacturer’s instructions. e absorbance at nm was
Evidence-Based Complementary and Alternative Medicine
measured on a microplate reader (BioTek SYNERGY ,
USA).
Blood was collected from the abdominal aorta and anti-
coagulated with citrate (.%, : , v/v). Platelet-rich plasma
(PRP) was obtained by centrifugation at rpm at ∘C
for min and the remaining blood was further centrifuged
at rpm at ∘C for min to prepare platelet-poor
plasma (PPP). e platelet concentration was adjusted to
4×10
8platelets/mL. e platelet agonist adenosine diphos-
phate disodium (Shanghai Institute of Biochemistry, Chinese
Academy of Sciences) ( mmol/L, 𝜇L) was used to stimulate
platelet aggregation. e level of platelet aggregation was
measured using an aggregometer (BS, Beijing) according
to the method reported by Born and Cross [].
2.6. Cell Culture, Hypoxia/Reoxygenation (H/R), and Drug
Tre atme nt. Primary cultures of neonatal rat cardiomyocytes
were prepared as previously described with some modica-
tions [,]. In brief, the hearts were removed and washed
with cold PBS. e atria and aorta were discarded. e
ventricles were minced with scissors into mm3fragments.
e tissue fragments were digested by gentle shaking at
∘C in PBS containing . g/L trypsin (Gibco) and . g/L
collagenase II (Gibco). e digestion was conducted for –
times, min each. e dispersed cells were incubated on a
mm culture dish for h at ∘Cinahumidiedincubator
with % CO2.enonadherentcellswereharvestedand
then seeded into gelatin-coated -well plates and incubated
in Dulbecco’s modied Eagle’s medium (DMEM) (Gibco)
with % newborn calf serum (TBDHY, Tianjin), penicillin
( U/mL), streptomycin ( U/mL), and -bromo--
deoxyuridine (. mmol/L, sigma), which was used to inhibit
cardiac broblasts growth.
Hypoxia/reoxygenation-induced cardiomyocytes injury
was performed as described previously []. Before hypoxia,
cardiomyocytes were washed three times with glucose-
free Tyrode’s solution (in mmol/L: NaCl ., KCl .,
NaH2PO4., NaHCO3., MgCl2., and CaCl2.).
Cells were incubated with glucose-free Tyrode’s solution
( mL/well) saturated with % N2and % CO2for min.
Cells were placed into a hypoxia chamber which was then
ventilated with % N2and % CO2for min and main-
tained at ∘C in a humidied incubator with % CO2
for h. Reoxygenation was accomplished by replacing the
glucose-free Tyrode’s solution with normal cell medium
under normoxic conditions. Reoxygenation time varied dep-
ending on the experimental objectives: h reoxygenation
was performed for measurement of LDH release, whereas
h reoxygenation was performed for ow cytometry assay
and measurements of cleaved caspase-, Akt, and p-Akt
expressions.
In the normal control group, cells were cultured with
Tyrode’s solution that contained .mmol/L glucose for h
andreoxygenatedwithDMEMforhorh.Inthetreat-
ment groups, diltiazem (positive control, nal concentration:
𝜇g/mL), citric acid, and L-malic acid (nal concentrations:
, , , 𝜇g/mL) were dissolved with dimethylsulfox-
ide (DMSO) and added, respectively, into the medium with
the ratio of : at the start of hypoxia and reoxygenation.
For the normal control group and model control group,
equivalent volumes of DMSO were added.
2.7. LDH Release. e hypoxia and reoxygenation super-
natants were collected. Aer h reoxygenation, cells were
lysed by freeze thawing in distilled water. LDH activities were
measured using the enzymatic reaction kinetics monitoring
method according to the manufacturer’s instructions.
e total LDH activity was obtained from adding LDH
activities in the hypoxia and reoxygenation supernatants and
the cell lysate together. e LDH release rate was calculated
by dividing the sum of LDH activities in the hypoxia and
reoxygenation supernatants into the total LDH activity.
2.8. Flow Cytometry Analysis. Apoptosis was assessed by
FITC-annexin V/propidium iodide apoptosis detection kit
according to the manufacturer’s protocol. Briey, at the end of
h reoxygenation, cells were harvested with trypsin (.%)
and centrifugation ( rpm for min). Cells were washed
twicewithcoldPBSandthenresuspendedin𝜇Lbinding
buer at a concentration of 1×10
5cells/mL. Cells were
incubated with 𝜇L FITC-annexin V and 𝜇Lpropidium
iodide (PI) for min in the dark at room temperature
(∘C). Samples were analyzed by ow cytometry (Epics Elite,
Beckman Coulter) immediately. Approximately cells
werecountedforeachsampleanddatawereanalyzedby
using Expo soware.
2.9. Western Blot Analysis. e expression levels of cleaved
caspase-, Akt, and p-Akt were measured by western blotting.
Cells were washed with prewarmed PBS and then lysed at
∘C with ice-cold RIPA lysis buer ( mM Tris (pH .),
mM NaCl, % Triton X-, % sodium deoxycholate,
.% SDS, mM PMSF, and phosphatase inhibitors mixture
(P, Applygen Technologies Inc.)) for min. Cell lysates
were then centrifuged at g at ∘C for min and protein
concentrations in the supernatants were determined by BCA
protein assay kit (Beyotime Biotechnology).
Samples with equivalent amounts of total protein ( 𝜇g)
were loaded and separated by % SDS-polyacrylamide gel
electrophoresis and transferred to polyvinylidene diuo-
ride (PVDF) membranes (Bio-Rad). e membranes were
blocked in % BSA for h and then incubated overnight at
∘Cwithprimaryantibody(rabbitanti-cleavedcaspase,
rabbit anti-Akt, and rabbit anti-p-Akt at : , : , and
: dilution, and mouse anti-𝛼-actin (Beijing Biosyn-
thesis Biotechnology, China) at : dilution). e mem-
branes were washed six times in ×Tris-buer saline-Tween
(TBST) buer and then incubated with horseradish-
peroxidase-(HRP-) conjugated goat anti-rabbit or mouse
secondary antibodies ( : dilution) for h at room tem-
perature. Aer excess antibodies were removed by washing,
bands were detected with an enhanced chemiluminescence
(ECL) system (ermo, USA) and visualized with the Chemi
Doc XRS+ gel documentation system (Bio-Rad, USA) and
analyzed by using Image lab . soware (Bio-Rad, USA). e
expression levels of 𝛼-actin served as an internal control for
protein loading.
Evidence-Based Complementary and Alternative Medicine
Sham Model
control
Diltiazem
16 mg/kg
Clopidogrel
13.5 mg/kg
Sham Model
control
Diltiazem
16 mg/kg
Clopidogrel
13.5 mg/kg
Citric acid
500 mg/kg
Citric acid
250 mg/kg
500 mg/kg 250 mg/kg
Red (ischemic) Dark (viable)
L-malic acidL-malic acid
(a)
(b)
F : A representative N-BT staining of infarct size. e normal myocardium was stained dark, and the ischemic area was stained red.
(a) Citric acid; (b) L-malic acid.
T : e eect of citric acid on myocardial ischemia/reperfusion injury in rats (𝑥±𝑠,n=).
Groups Dosage (mg/kg) Infarction of the ventricle (%) Infarction of the heart (%) Infarction area (mm) Infarction weight (g)
Sham — 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.000 ± 0.000
Model control — 17.30 ± 3.58## 11.98 ± 2.20## 49.75 ± 9.14## 0.086 ± 0.017##
Diltiazem 8.23 ± 2.50∗∗ 5.33 ± 1.11∗∗ 21.90 ± 4.78∗∗ 0.038 ± 0.007∗∗
Clopidogrel . 11.35 ± 2.71∗∗ 8.52 ± 1.77∗∗ 35.17 ± 9.74∗∗ 0.064 ± 0.015∗
Citric acid 12.16 ± 4.27∗∗ 8.80 ± 3.41∗∗ 36.18 ± 15.23∗∗ 0.064 ± 0.026∗
11.80 ± 3.67∗∗ 8.83 ± 3.09∗∗ 33.97 ± 10.99∗∗ 0.063 ± 0.022∗∗
𝑃 < 0.01 versus sham, ∗∗𝑃 < 0.01,∗𝑃 < 0.05 versus model control.
2.10. Statistical Analysis. All data were presented as the
mean ±SD. e data analyses were performed using one-way
ANOVA analysis followed by Student-Newman-Keuls test for
multiple comparisons. In all cases, values of 𝑃 < 0.05 were
considered statistically signicant.
3. Results
3.1. Eects of Citric Acid and L-Malic Acid on Myocardial
Infarct Size. As illustrated in Figure (a) and Table ,no
myocardial infarction was observed in the sham group,
while myocardial ischemia/reperfusion resulted in signicant
myocardial infarcts (𝑃 < 0.01). Diltiazem and clopidogrel,
which were used as positive controls, signicantly reduced
infarction percentage of the ventricle, infarction percentage
of the heart, infarction area, and infarction weight, as com-
paredwiththemodelcontrol(𝑃 < 0.01 or 𝑃 < 0.05). Com-
pared with the model control group, treatments with citric
acid at the doses of mg/kg and mg/kg signicantly
reduced infarction percentage of the ventricle, infarction
percentage of the heart, infarction area, and infarction weight
(𝑃 < 0.01 or 𝑃 < 0.05).
A similar result was shown in Figure (b) and Table .
Myocardial ischemia/reperfusion resulted in substantial
myocardial infarcts, which were signicantly reduced by
treatments with diltiazem and clopidogrel (𝑃 < 0.01 or 𝑃<
0.05). Compared with the model control group, treatment
with L-malic acid at the dose of mg/kg signicantly
decreased infarction percentage of the ventricle, infarction
percentageoftheheart,infarctionarea,andinfarctionweight
(𝑃 < 0.01), and treatment with L-malic acid at the dose
of mg/kg signicantly decreased infarction percentage of
the ventricle and infarction area (𝑃 < 0.01 and 𝑃 < 0.05,
resp.) but had only a tendency to reduce infarction percentage
oftheheartandinfarctionweight(𝑃 = 0.056 and 𝑃 = 0.095,
resp.).
3.2. Eects of Citric Acid and L-Malic Acid on TNF-𝛼Produc-
tion following Myocardial Ischemia/Reperfusion. Figure (a)
showed that myocardial ischemia/reperfusion injury signif-
icantly increased the level of serum TNF-𝛼compared with
the sham group (26.71 ± 6.44 versus 11.84 ± 1.67 pg/mL, 𝑃<
0.05). Compared with the model control group, pretreatment
with clopidogrel signicantly reduced serum TNF-𝛼level by
.% (14.51 ± 3.02pg/mL, 𝑃 < 0.01), and pretreatments
with citric acid at the doses of mg/kg and mg/kg
signicantly reduced serum TNF-𝛼levels by .% (22.66 ±
Evidence-Based Complementary and Alternative Medicine
T : e eect of L-malic acid on myocardial ischemia/reperfusion injury in rats (𝑥±𝑠,𝑛=10).
Groups Dosage (mg/kg) Infarction of the ventricle (%) Infarction of the heart (%) Infarction area (mm) Infarction weight (g)
Sham — 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.000 ± 0.000
Model control — 9.03 ± 3.32## 6.00 ± 1.67## 32.01 ± 11.84## 0.051 ± 0.014##
Diltiazem 6.86 ± 1.76∗∗ 4.68 ± 1.39∗25.11 ± 6.48∗0.041 ± 0.012∗
Clopidogrel . 6.51 ± 1.58∗∗ 4.75 ± 1.19∗23.67 ± 5.46∗0.038 ± 0.010∗
L-malic acid 6.71 ± 1.23∗∗ 4.98 ± 0.92 25.37 ± 4.55∗0.043 ± 0.009
6.22 ± 1.15∗∗ 4.48 ± 0.93∗∗ 22.70 ± 4.26∗∗ 0.036 ± 0.007∗∗
𝑃 < 0.01 versus sham, ∗∗𝑃 < 0.01,∗𝑃 < 0.05 versus model control.
##
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
Groups
Sham Model control
Citric acid 500 mg/kg
Citric acid 250 mg/kg
∗∗
∗∗
∗
Clopidogrel 13.5 mg/kg
TNF-𝛼(pg/mL)
(a)
##
0.0
5.0
10.0
15.0
20.0
25.0
Groups
Sham Model control
L-malic acid 500 mg/kg
L-malic acid 250 mg/kg
Clopidogrel 13.5 mg/kg
TNF-𝛼(pg/mL)
∗∗
∗
(b)
F : Eects of citric acid (a) and L-malic acid (b) on serum levels of TNF-𝛼following myocardial ischemia/reperfusion. Data are shown
as mean ±SD. ##𝑃< 0.01 versus sham, ∗∗ 𝑃< 0.01,∗𝑃< 0.05 versus model control (𝑛=10).
5.22pg/mL, 𝑃 < 0.05) and .% (20.49 ± 2.71pg/mL, 𝑃<
0.01), respectively.
Similarly, Figure (b) showed that the level of serum TNF-
𝛼in the model control group was signicantly increased
(16.42 ± 7.27 versus 9.85 ± 2.25 pg/mL in the sham group,
𝑃 < 0.05). Compared with the model control group, pre-
treatment with clopidogrel reduced serum TNF-𝛼level by
.% (11.36± 3.73pg/mL, 𝑃 < 0.05), and pretreatment with
L-malic acid at the dose of mg/kg had only a tendency
to decrease serum TNF-𝛼level (12.98 ± 4.63pg/mL, 𝑃=
0.129), while pretreatment with L-malic acid at the dose of
mg/kg signicantly reduced serum TNF-𝛼level by .%
(10.20 ± 1.50 pg/mL, 𝑃 < 0.01).
3.3. Eects of Citric Acid and L-Malic Acid on Platelet Aggre-
gation Induced by ADP following Myocardial Ischemia/Repe-
rfusion. We measured the eects of citric acid and L-malic
acid on platelet aggregation induced by one of the classical
endogenous agonists ADP. As shown in Figure (a),com-
pared with the sham group, myocardial ischemia/reperfusion
signicantly increased platelet aggregation rate induced by
ADP (57.53 ± 7.47%versus.81 ± 6.18%, 𝑃 < 0.05).
Compared with the model control group, pretreatment with
clopidogrel signicantly reduced platelet aggregation rate
(2.12±3.44%, 𝑃 < 0.01), and pretreatments with citric acid at
the doses of mg/kg and mg/kg signicantly decreased
platelet aggregation rate (35.19 ± 13.29%and27.50 ±14.08%,
resp., 𝑃 < 0.01 each).
A similar result was shown in Figure (b),theplatelet
aggregation rate in the model control group was signicantly
increased (66.43 ± 8.66%versus.06 ± 5.27%inthesham
group, 𝑃 < 0.05). Compared with the model control group,
the platelet aggregation rate for the clopidogrel group was
3.36 ± 4.15%(𝑃 < 0.01), and that for L-malic acid at the
doses of mg/kg and mg/kg groups was 47.02±17.09%
(𝑃 < 0.01)and57.58 ± 8.09%(𝑃 = 0.149), respectively.
3.4. Eects of Citric Acid and L-Malic Acid on H/R-Induced
Cardiomyocyte Necrosis. LDH leakage from cells was widely
used as a reliable marker of cellular injury. e degree of
LDH release was closely related to cardiomyocyte necrosis
[,]. us, we explored the protective eects of citric acid
and L-malic acid on H/R-induced cardiomyocyte injury in
vitro by detecting LDH release. Figure (a) showed that aer
cardiomyocytes were subjected to h hypoxia followed by
h reoxygenation, a signicant LDH release was induced
(40.76 ± 2.88%versus14.57 ± 0.96%inthenormalcontrol
group, 𝑃 < 0.01), which were signicantly inhibited by
diltiazem (23.25 ± 2.61%, 𝑃 < 0.01) and citric acid at the
concentration of 𝜇g/mL (31.07 ± 5.54%, 𝑃 < 0.01).
A similar result was shown in Figure (b),theLDH
releaserateinthemodelcontrolgroupwassignicantly
Evidence-Based Complementary and Alternative Medicine
#
0.0
10.0
20.0
30.0
40.0
50.0
60.0
70.0
Platelet aggregation (%)
Groups
Sham Model control
Citric acid 500 mg/kg
Citric acid 250 mg/kg
∗∗
∗∗
∗∗
Clopidogrel 13.5 mg/kg
(a)
#
0.0
10.0
20.0
30.0
40.0
50.0
60.0
70.0
80.0
Groups
Platelet aggregation (%)
∗∗
∗∗
Sham Model control
L-malic acid 500 mg/kg
L-malic acid 250 mg/kg
Clopidogrel 13.5 mg/kg
(b)
F : Eects of citric acid (a) and L-malic acid (b) on platelet aggregation induced by ADP following myocardial ischemi-reperfusion.
Data are shown as mean ±SD. #𝑃< 0.05 versus sham, ∗∗𝑃< 0.01 versus model control (𝑛=10).
##
0.0
10.0
20.0
30.0
40.0
50.0
LDH leakage rate (%)
∗∗
∗∗
Groups
Normal control
Diltiazem 45 𝜇g/mL
Citric acid 100 𝜇g/mL
Normal control
Citric acid 200 𝜇g/mL
Citric acid 50 𝜇g/mL
(a)
##
0.0
10.0
20.0
30.0
40.0
50.0
60.0
∗∗
Groups
Normal control
Diltiazem 45 𝜇g/mL
Normal control
LDH leakage rate (%)
∗
L-malic acid 100 𝜇g/mL
L-malic acid 200 𝜇g/mL
L-malic acid 50 𝜇g/mL
(b)
F : Eects of citric acid (a) and L-malic acid (b) on LDH release. Cardiomyocytes were subjected to h hypoxia followed by h
reoxygenation with or without treatment. LDH activities in the hypoxia and the reoxygenation media and in the cell lysates were measured.
Data are shown as mean ±SD. ##𝑃< 0.01 versus normal control, ∗∗𝑃< 0.01,∗𝑃< 0.05 versus model control (𝑛=3).
increased (45.31 ± 3.00%versus11.23 ± 0.86%innormal
control group, 𝑃 < 0.01). Compared with the model
control group, the LDH release rate for diltiazem group was
19.12 ± 0.57%(𝑃 < 0.01),andthatforL-malicacidatthe
concentration of 𝜇g/mL group was 40.69 ± 4.03%(𝑃<
0.05).
3.5. Eects of Citric Acid and L-Malic Acid on H/R-Induced
Cardiomyocyte Apoptosis. Based on the previous results that
treatments with citric acid and L-malic acid below con-
centration of 𝜇g/mL could not decrease LDH release,
we chose to use citric acid and L-malic acid at concen-
trations of 𝜇g/mL and 𝜇g/mL to observe whether
citric acid and L-malic acid could decrease H/R-induced
cardiomyocyte apoptosis. As data shown in Figure (a),aer
cardiomyocytes were subjected to h hypoxia followed by
h reoxygenation injury, the number of apoptotic cells was
signicantly increased as compared with the normal control
group (25.45 ± 1.81%versus11.48 ± 2.74%, 𝑃 < 0.01).
In contrast, treatments with citric acid at concentrations of
𝜇g/mL and 𝜇g/mL reduced the number of apoptotic
cells to 19.43 ± 1.69%(𝑃 < 0.01)and22.70 ± 3.47%(𝑃=
0.179), respectively.
Similarly, Figure (b) showed that the number of apop-
totic cells in the model control group was signicantly
increased (22.13 ± 1.69%versus15.65 ± 1.34%inthenormal
control group, 𝑃 < 0.01), which was signicantly reduced by
treatments with L-malic acid at concentrations of 𝜇g/mL
(18.63 ± 3.17%, 𝑃 < 0.05)and𝜇g/mL (16.70 ± 0.62%,
𝑃 < 0.01), respectively.
Evidence-Based Complementary and Alternative Medicine
B1
0.6%
B3
88.5%
B4
9.6%
B2
1.3%
100101102103
Annexin-FITC
103
102
101
100
PI
B1
0.4%
B3
71.4%
B4
25.6%
B2
2.5%
100101102103
Annexin-FITC
103
102
101
100
PI
B1
0.9%
B3
81.1%
B4
15.4%
B2
2.6%
100101102103
Annexin-FITC
103
102
101
100
PI
B1
0.5%
B3
80.8%
B4
16.7%
B2
2.0%
100101102103
Annexin-FITC
103
102
101
100
PI
##
0.0
5.0
10.0
15.0
20.0
25.0
30.0
Groups
Normal control
Normal control
Model control
Model control
∗∗
Apoptotic rate (%)
Citric acid 200 𝜇g/mL
Citric acid 200 𝜇g/mL
Citric acid 400 𝜇g/mL
Citric acid 400 𝜇g/mL
(a)
##
0.0
5.0
10.0
15.0
20.0
25.0
30.0
Groups
Normal control Model control
∗∗
∗
Apoptotic rate (%)
L-malic acid 400
𝜇g
/mL L-malic acid 200 𝜇g/mL
B1
0.4%
B3
84.9%
B4
12.7%
B2
2.0%
100101102103
Annexin-FITC
103
102
101
100
PI
B1
0.3%
B3
76.0%
B4
21.4%
B2
2.2%
100101102103
Annexin-FITC
103
102
101
100
PI
B1
0.5%
B3
84.2%
B4
12.8%
B2
2.5%
100101102103
Annexin-FITC
103
102
101
100
PI
B1
0.4%
B3
83.1%
B4
14.2%
B2
2.3%
100101102103
Annexin-FITC
103
102
101
100
PI
Normal control Model control L-malic acid 400 𝜇g/mL L-malic acid 200 𝜇g/mL
(b)
F : Eects of citric acid (a) and L-malic acid (b) on H/R-induced cardiomyocyte apoptosis. Cardiomyocytes were subjected to h
hypoxia and h reoxygenation in the presence or absence of citric acid or L-malic acid. Data are shown as mean ±SD. ##𝑃< 0.01 versus
normal control, ∗∗𝑃< 0.01,∗𝑃< 0.05 versus model control (𝑛=3).
Evidence-Based Complementary and Alternative Medicine
##
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
Groups
∗∗
∗
∗∗
Cleaved caspase-3 (fold increase)
Normal control
L-malic acid 400 𝜇g/mL
Citric acid 400 𝜇g/mL
Model control
L-malic acid 200 𝜇g/mL
Citric acid 200 𝜇g/mL
Normal L-malic acid (𝜇g/mL) Citric acid (𝜇g/mL)
Model
400 200 400 200
𝛼-actin
Cleaved
caspase-3
F : Eects of citric acid and L-malic acid on expression
levels of cleaved caspase- (fold increase relative to normal control
levels) aer cardiomyocytes subjected to h hypoxia followed by h
reoxygenation. Data are shown as mean ±SD. ##𝑃< 0.01 versus
normal control, ∗∗𝑃< 0.01,∗𝑃< 0.05 versus model control.
Results are representative of three independent experiments.
3.6. Eects of Citric Acid and L-Malic Acid on Expression of
Cleaved Caspase-3. Next we investigated the eects of citric
acid and L-malic acid on expression levels of cleaved caspase-
, the activated form of caspase-. As shown in Figure ,
western blot analysis revealed that the expression of cleaved
caspase- was signicantly upregulated (.-fold, 𝑃 < 0.01)
aer cardiomyocytes were subjected to h hypoxia followed
by h reoxygenation, which was signicantly downregulated
by treatments with citric acid at the concentrations of
𝜇g/mL (.%, 𝑃 < 0.05)and𝜇g/mL (.%, 𝑃<
0.05) and treatments with L-malic acid at concentrations of
𝜇g/mL (.%, 𝑃 < 0.05)and𝜇g/mL (.%, 𝑃<
0.01), respectively.
3.7. Eects of Citric Acid and L-Malic Acid on Expression
Levels of Akt and p-Akt. e PI K/Akt pathway plays a
critical role in survival aer myocardial ischemia/reperfusion
injury. Phosphorylation of Akt S represents its maximal
activation [,]. To determine whether Akt was involved in
citric acid and L-malic acid protection from cardiomyocyte
injury, we further detected the expressions of Akt and
phospho-Akt (Ser ). As illustrated in Figure ,western
blotting results showed that total Akt was comparable in all
groups. e densities of phosphorylated Akt were normalized
against total Akt. We found that H/R-induced cardiomyocyte
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
Groups
∗
∗
Normal control
L-malic acid 400 𝜇g/mL
Citric acid 400 𝜇g/mL
Model control
L-malic acid 200 𝜇g/mL
Citric acid 200 𝜇g/mL
Normal L-malic acid (𝜇g/mL)Citric acid (𝜇g/mL)
Model
400 200400 200
Tot al Ak t Phospho-Akt
Expressions of total Akt and P-Akt
(fold increase)
P-Akt
T-Akt
𝛼-actin
F : Citric acid and L-malic acid activate PI/K and phospho-
rylation of Akt. A representative western blot analysis of total Akt
and phosphorylation of Akt at Ser aer cardiomyocytes were
subjected to h hypoxia followed by h reoxygenation. Data are
shown as mean ±SD. ∗𝑃< 0.05 versus model control. Results are
representative of three independent experiments.
injury by itself resulted in a .-fold increase in Akt phos-
phorylation, while treatments with both citric acid at the
concentration of 𝜇g/mL and L-malic acid at the con-
centration of 𝜇g/mL further signicantly upregulated the
expression levels of phosphorylated Akt aer cardiomyocytes
hypoxia/reoxygenation injury compared with the model
control group (.-fold and .-fold, resp., 𝑃 < 0.05
each). Treatments with both citric acid at the concentration
of 𝜇g/mL and L-malic acid at the concentration of
𝜇g/mL had a tendency to increase the expression levels
of phosphorylated Akt (.% and .%, resp., 𝑃 = 0.763
and 𝑃 = 0.337, resp.), without signicant dierences.
4. Discussion
In the present study, we reported for the rst time the
in vivo data demonstrating that pretreatments with both
citric acid and L-malic acid signicantly ameliorated the I/R-
inducedcardiacinjury,includingreducedmyocardialinfarct
size, decreased inammatory cytokine TNF-𝛼activity, and
inhibited ADP-induced platelet aggregation. Furthermore,
in vitro experiments revealed that both citric acid and L-
malic acid protected cardiomyocyte damage from necrosis
and apoptosis during cardiomyocyte hypoxia/reoxygenation
injury possibly via a mechanism involving PIK/Akt survival
pathway.
Evidence-Based Complementary and Alternative Medicine
In recent years, traditional Chinese medicine has been
greatly developed in many countries due to its high quality
and safety [–], and considerable attention has focused
on the material basis of Chinese medicine studies. e
material basis of Fructus Choerospondiatis responsible for
its cardioprotective eects has been always considered to
be avonoids (mainly quercetin). However, our data in vivo
demonstrated that both citric acid (, mg/kg) and
quercetin (, mg/kg) signicantly ameliorated the I/R-
induced cardiac injury (data unpublished), and the in vitro
experiments conrmed that both organic acids (citric acid,
L-malic acid, succinic acid, and tartaric acid; concentra-
tion: 𝜇g/mL) and avonoids (quercetin and kaempferol;
concentrations: ., , , and 𝜇g/mL) signicantly
decreasedLDHreleaserateofcardiomyocytesinjuredby
hypoxia/reoxygenation [,]. Although the dosage of
organic acids used in these studies was ∼ times higher
than that of avonoids, the content of total organic acids in
Fructus Choerospondiatis was nearly times higher than
that of total avonoids. erefore, our results still furnish
strong evidence that organic acids may also be the major
active ingredients of Fructus Choerospondiatis responsible for
its cardioprotective eects.
e extent of myocardial damage is closely related to
prognosis. erefore, determination of infarct size is the
strongest determinant of prognosis of ischemic heart disease
[]. e results showed that pretreatments with both citric
acid and L-malic acid signicantly reduced I/R-induced
myocardial infarct size and thus protected the infarcted
myocardium.
Inammatory responses and platelet aggregation have
been implicated in myocardial ischemia/reperfusion injury
[]. Within minutes aer reperfusion, inammatory cas-
cade is triggered and copious amounts of proinammatory
cytokinessuchasTNF-𝛼,IL-𝛽, IL-, and IL- are pro-
duced and released []. ese proinammatory cytokines
(particularly TNF-𝛼), as important factors in cardiac dys-
function, activate neutrophils and endothelial cells and
aggravate myocardial ischemia/reperfusion injury [,].
Platelets play a critical role in the process of myocardial
ischemia/reperfusion injury. Aer reperfusion, platelets are
immediately activated and increased, and platelet aggrega-
bility will aggravate myocardial ischemia/reperfusion injury
in turn, which may be related to endothelial dysfunction
and platelet-derived p-selectin, and so forth []. e results
showed that both citric acid and L-malic acid decreased
TNF-𝛼level and inhibited platelet aggregation on myocar-
dial ischemia/reperfusion injury. ese data in vivo pro-
vided direct evidence that organic acids protected ischemia
myocardium may be partly due to inhibition of inammation
and platelet aggregation.
Cardiomyocyte necrosis and apoptosis are the major
contributors to myocardial ischemia/reperfusion injury [].
Cardiomyocyte loss, caused by both necrosis and apopto-
sis, is the main feature of myocardial ischemia/reperfusion
injury []. Necrosis and apoptosis are two distinct types
of cell death with dierent characteristics. Necrosis leads
to membrane lysis, release of cellular contents, and result-
ing inammation, while apoptosis is characterized by cell
shrinkage, membrane blebbing, and nuclear condensation
and degradation []. Necrotic cells are mainly found in the
central zone of the infarct, while apoptotic cells are more
apparent at the marginal zone []. It may be benecial for
attenuating necrosis and apoptosis to prevent cardiomyocyte
loss caused by myocardial ischemia/reperfusion injury. us,
aer an initial investigation of the eects of citric acid and
L-malic acid on myocardial ischemia/reperfusion injury in
in vivo rat model, we further observed their cardioprotective
eects in cellular level. e concentrations of citric acid and
L-malic acid ( 𝜇g/mL and 𝜇g/mL) used in this study
had been evaluated on cytotoxicity, as determined by MTT
assay. ere were no signicant dierences between citric
acid or L-malic acid at the concentrations below 𝜇g/mL
and the control group []. LDH is a stable cytosolic enzyme
present in mammalian cells and LDH release is an indication
of cell membrane integrity. e amount of LDH released
from cells is proportional to the extent of membrane damage
and cell necrosis []. e data showed that H/R injury
induced signicant LDH release, but treatments with citric
acid at the concentration of 𝜇g/mL and L-malic acid at
the concentration of 𝜇g/mL could signicantly reduce
cardiomyocyte LDH release rate.
Furthermore, we studied the eects of citric acid and L-
malic acid on hypoxia/reoxygenation-induced apoptosis by
ow cytometry analysis. e results showed that H/R injury
signicantly increased the number of apoptotic cardiomy-
ocytes, while treatments with citric acid at the concentration
of 𝜇g/mL or L-malic acid at the concentrations of
𝜇g/mL and 𝜇g/mL signicantly reduced the number
of apoptotic cells. e cleavage of caspase- is oen identied
as the important step in the apoptotic signaling pathway
activation process and it is considered to be a potential
molecular therapeutic target for preventing cardiomyocyte
apoptosis []. We next investigated the expression of
cleaved caspase- and found that the cleaved caspase- was
signicantly upregulated by hypoxia/reoxygenation-induced
cardiomyocyte injury while signicantly downregulated by
treatments with citric acid at the concentrations of 𝜇g/mL
and 𝜇g/mL or L-malic acid at the concentrations of
𝜇g/mL and 𝜇g/mL relative to the model control
group.
Akt is a potent cell survival factor and an important
downstream kinase of PI K. e phosphor ylation and activa-
tion of Akt play a pivotal role in myocardial ischemia/reper-
fusion injury []. Considerable evidence suggests that the
activation of Akt reduced myocardial infarct size [,]. To
investigate whether Akt is involved in citric acid and L-malic
acid-induced cardioprotection, we evaluated the expression
of Akt and its activated, phosphorylated form (phospho-Akt)
at Ser aer hypoxia/reoxygenation-induced cardiomy-
ocyte injury. Our results showed that treatments with citric
acid and L-malic acid signicantly upregulated the expression
of phosphorylated Akt. All data in vitro concluded that the
cardioprotection of citric acid and L-malic acid contributed
to preventing cardiomyocyte from necrosis and apoptosis,
which may have the PIK/Akt sur vival pathway involved.
In conclusion, the present study demonstrates that
citric acid and L-malic acid have protective eects on
Evidence-Based Complementary and Alternative Medicine
myocardial ischemia/reperfusion injury; the underlying
mechanism may be associated with their anti-inammatory,
anti-platelet aggregation and direct cardiomyocyte protective
eects. Based on these ndings, we concluded that organic
acids may also be the major active ingredients of Fructus
Choerospondiatis responsible for its cardioprotective eects
but not only avonoids now.
Conflict of Interests
e authors declare that there is no conict of interests.
Acknowledgments
is research was supported by grants from the National
Natural Science Foundation of China (Grant nos. ;
) and the National Science & Technology Major
ProjectofChina(Grantnos.zx--;
zx -).
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