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Cardioprotective Effect of Monoammonium Glycyrrhizinate Injection Against Myocardial Ischemic Injury in vivo and in vitro: Involvement of Inhibiting Oxidative Stress and Regulating Ca2+ Homeostasis by L-Type Calcium Channels

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Purpose: Monoammonium glycyrrhizinate (MAG) is an aglycone of glycyrrhizin that is found in licorice and is often used clinically as an injection to treat liver diseases. However, the effect of MAG injection on cardiac function and its possible cellular mechanisms remain unclear. We explored the protective effects of MAG against myocardial ischemic injury (MII) induced by isoproterenol (ISO), as well as the cellular mechanisms via molecular biology techniques and patch-clamp recording. Methods: A rat model of myocardial ischemia injury was induced by administering ISO (85 mg/kg) subcutaneously for 2 consecutive days. ECG, cardiac functional parameters, CK and LDH levels, SOD and GSH activities, MDA concentration, histological myocardium inspection, mitochondria ultrastructure changes, intracellular calcium concentrations were observed. Influences of MAG on ICa-L and contraction in isolated rat myocytes were observed by the patch-clamp technique. Results: MAG reduced damage, improved cardiac morphology, inhibited oxidative stress, decreased the generation of reactive oxygen species, and decreased intracellular Ca2+ concentration. Exposure of the rats' ventricular myocytes to MAG resulted in a concentration-dependent reduction in L-type calcium currents (ICa-L). MAG reduced ICa-L in a consistent and time-dependent fashion with a semi-maximal prohibitive concentration of MAG of 14 μM. MAG also shifted the I-V curve of ICa-L upwards and moved the activation and inactivation curves of ICa-L to the left. Conclusion: The findings indicate that MAG injection exerts a protective influence on ISO-induced MII by inhibiting oxidative stress and regulating Ca2+ homeostasis by ICa-L.
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ORIGINAL RESEARCH
Cardioprotective Effect of Monoammonium
Glycyrrhizinate Injection Against Myocardial
Ischemic Injury in vivo and in vitro: Involvement of
Inhibiting Oxidative Stress and Regulating Ca
2+
Homeostasis by L-Type Calcium Channels
This article was published in the following Dove Press journal:
Drug Design, Development and Therapy
Zhifeng Zhao,
1,
*
Miaomiao Liu,
1,
*
Yuanyuan Zhang,
1
Yingran Liang,
1
Donglai Ma,
1
Hongfang Wang,
1
Zhihong Ma,
2
Shengjiang Guan,
2
Zhonglin Wu,
3
Xi Chu,
3
Yue Lin,
2
Li Chu
1,4
1
School of Pharmacy, Hebei University of
Chinese Medicine, Shijiazhuang 050200,
Hebei, Peoples Republic of China;
2
School of Basic Medicine, Hebei
University of Chinese Medicine,
Shijiazhuang 050200, Hebei, Peoples
Republic of China;
3
The Fourth Hospital
of Hebei Medical University, Shijiazhuang
050011, Hebei, Peoples Republic of
China;
4
Hebei Key Laboratory of
Integrative Medicine on Liver-Kidney
Patterns, Shijiazhuang 050200, Hebei,
Peoples Republic of China
*These authors contributed equally to
this work
Purpose: Monoammonium glycyrrhizinate (MAG) is an aglycone of glycyrrhizin that is
found in licorice and is often used clinically as an injection to treat liver diseases. However,
the effect of MAG injection on cardiac function and its possible cellular mechanisms remain
unclear. We explored the protective effects of MAG against myocardial ischemic injury
(MII) induced by isoproterenol (ISO), as well as the cellular mechanisms via molecular
biology techniques and patch-clamp recording.
Methods: A rat model of myocardial ischemia injury was induced by administering ISO (85
mg/kg) subcutaneously for 2 consecutive days. ECG, cardiac functional parameters, CK and
LDH levels, SOD and GSH activities, MDA concentration, histological myocardium inspec-
tion, mitochondria ultrastructure changes, intracellular calcium concentrations were
observed. Inuences of MAG on I
Ca-L
and contraction in isolated rat myocytes were
observed by the patch-clamp technique.
Results: MAG reduced damage, improved cardiac morphology, inhibited oxidative stress,
decreased the generation of reactive oxygen species, and decreased intracellular Ca
2+
con-
centration. Exposure of the ratsventricular myocytes to MAG resulted in a concentration-
dependent reduction in L-type calcium currents (I
Ca-L
). MAG reduced I
Ca-L
in a consistent
and time-dependent fashion with a semi-maximal prohibitive concentration of MAG of 14
μM. MAG also shifted the I-V curve of I
Ca-L
upwards and moved the activation and
inactivation curves of I
Ca-L
to the left.
Conclusion: The ndings indicate that MAG injection exerts a protective inuence on ISO-
induced MII by inhibiting oxidative stress and regulating Ca
2+
homeostasis by I
Ca-L
.
Keywords: cardiopretection, reactive oxygen species, calcium inux, isoproterenol, calcium
concentration
Introduction
Licorice is used as an herbal medicine and is native to East Asia and Southern Europe.
It has benecial applications in both medicine and confections
1
and is obtained from
Glycyrrhiza uralensis Fisch (Glycyrrhiza glabra L.).
2
Monoammonium glycyrrhizi-
nate (MAG) (Figure 1) is an aglycone of glycyrrhizin that is derived from licorice, and
modern medical research has shown that this substance is the main bioactive
Correspondence: Li Chu
School of Pharmacy, Hebei University of
Chinese Medicine, Shijiazhuang 050200,
Hebei, Peoples Republic of China
Email chuli0614@126.com
Yue Lin
School of Basic Medicine, Hebei
University of Chinese Medicine,
Shijiazhuang 050200, Hebei, Peoples
Republic of China
Tel/Fax +86 311 89926718
Email yuelin8992@126.com
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component of the plant. Many studies have focused on gly-
cyrrhizic acid and its derivatives, which have many biologi-
cal functions,
3,4
such as liver detoxication effects
5,6
and
antiallergic activities.
7,8
MAG has also been used clinically
as an injection to treat liver diseases.
9,10
Oxidative stress is a process that causes oxidative
damage through imbalance between the production and
elimination of oxygen free radicals in the body or cells,
which results in the accumulation of reactive oxygen spe-
cies (ROS).
11
In great quantities, free radicals can cause
tissue and cell damage through a series of peroxidation
reactions. Changes in malondialdehyde (MDA), a lipid
peroxidation product, can indirectly reect the metabolism
of oxygen free radicals in vivo, and its level reects the
severity of free radical attack on cells.
12
Superoxide dismutase (SOD) is a specic scavenger that
has great signicance in the activities of the body. As an
important scavenger of oxygen free radicals in vivo, it is an
important antioxidant enzyme and can protect cells from
damage. The level of SOD can indirectly reect the ability
ofthebodytoremovefreeradicals.
13
The production and
removal of free radicals occur in a dynamic equilibrium state
so that they do not easily cause tissue damage.
The interaction of free radicals and membrane-lipid
unsaturated fatty acids leads to lipid peroxidation, which
causes an imbalance in the ratio of membrane unsaturated
fatty acid to protein.
14
This interaction also decreases the
liquidity, uidity, and permeability of cell membranes and
organelle membranes, increases the inux of Ca
2+
and
indirectly inhibits the function of membrane protein.
These phenomena lead to increased concentrations of
cytoplasmic Ca
2+
cell swelling, and calcium overload.
15
Isoproterenol (ISO) is a synthetic catecholamine, and
its administration causes severe stress in the myocardium
due to the activation of the adrenergic system and other
neurohumoral systems, which lead to increases in L-type
Ca
2+
channel (LTCC) activity.
16
Ischemic heart disease
(IHD) is the most widely cited reason for morbidity and
Figure 1 Chemical structure of MAG.
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disability in the modern world. Heart failure, hypertension,
and ischemia from atherosclerosis partly result from
changes in intracellular Ca
2+
Mitochondrial permeability
transition pores (mPTPs) may be involved in the release of
mitochondrial components during cell death and play an
important role in cell survival and apoptosis.
17
Calcium
overload might lead to the opening of mPTPs, plasma-
lemma rupture, and even cell death.
15
LTCCs are the main
path of inux for Ca
2+
and are important for the treatment
of ischemic cardiac disease.
18
MAG injection has anti-inammatory, anti-allergic,
and membrane-protective effects. Thus, it has been used
clinically for immunoregulation, the promotion of biliru-
bin metabolism, the inhibition of viral hepatitis, the
improvement of liver function, and as an adjuvant treat-
ment for radiotherapy and chemotherapy when treating
cancer.
10
However, the cardioprotective effects of MAG
injection have not been proven. We speculate that MAG
exerts a myocardial protective effect by improving oxida-
tive stress and reducing cytoplasmic Ca
2+
concentration by
LTCCs.
In the present study, we evaluated the effects of MAG
injection on LTCC current (I
Ca-L
) in myocardial cells from
rats to elaborate the underlying cellular mechanisms of its
protective effects against myocardial ischemic injury
(MII). To this end, we examined the oxidative response
by the production of ROS, MDA, and SOD, detected
changes in cytoplasmic Ca
2+
concentration, and used
patch-clamp techniques to study the inuence of MAG
injection. The ndings could contribute to efforts to
improve the efcacy of MAG injection in clinical
treatments.
Materials and Methods
Materials
MAG injections were purchased from Yuanye Bio-
Technology Co., Ltd. (Shanghai, China). ISO was purchased
from Amylet Scientic Inc. (Michigan, USA). Verapamil
(Ver) was purchased from HeFeng Pharmaceutical Co., Ltd.
(Shanghai, China). Other reagents were purchased from
Sigma-Aldrich (St. Louis, MO, USA). Every solvent used
in the experiments was of analytical purity.
Animals
Male adult Sprague-Dawley rats were purchased from the
Experimental Animal Center of Hebei Medical University
and housed in standard growth conditions (2024 degrees
celsius, 4060% relative humidity, 12-h light-dark cycle).
The experiments began after at least a 7 days of adaptation
to the laboratory environment. All experimental and ani-
mal handling procedures were approved by the Ethics
Committee for Animal Experiments of Hebei University
of Chinese Medicine (approval number: DWLL2018028;
approval date: December 20, 2018) and conformed to the
National Institutes of Health Guidelines for the Care and
Use of Laboratory Animals.
Induction of MII
Forty adult male SD rats (200240 g) were kept at 2024
degrees celsius, with free intake of water and food. After 1
week of adaptive feeding, all animals were randomly
allocated to a control (Con), ISO, MAG, or verapamil
(Ver) group (n = 10 in each group). The MAG group and
Ver group were respectively given MAG (30 mg/kg/d) and
Ver (2 mg/kg/d), while the Con group and ISO group were
injected with puried water. All injections were intraper-
itoneal. After 1 week of continuous treatment with these
methods, ISO was injected subcutaneously (85 mg/kg) for
two consecutive days in all groups except for the Con
group. All experiments were approved by the Ethics
Committee of Hebei University of Chinese Medicine.
Acquisition of Electrocardiogram (ECG)
and Cardiac Functional Parameters
ECG recordings of J-point elevation and heart rate were
obtained from the animals using a BL-420S biological func-
tion experimental system. Anesthesia was performed by
intraperitoneal injection of ethyl carbamate (1.0 g/kg), and
ECG was performed at 30 min after the last injection of ISO
or a placebo. Three needle electrodes were connected to the
left front leg and both hind legs of the animals.
Catheterization of the left ventricle (LV) was per-
formed on anesthetized rats after nishing the nal injec-
tion of ISO. A miniature pressure transducer (Chengdu
instrument Co., Chengdu, China) was inserted into the
LV via the right carotid artery and advanced into the LV
under continuous monitoring of the pressure waveform.
Pressure signals were digitized and recorded with the BL-
420S. The heart rate (HR), left ventricular systolic pres-
sure (LVSP), left ventricular diastolic pressure (LVDP),
and the rst derivatives of left intraventricular pressure
(rate of pressure development +dP/dt
max
and rate of pres-
sure decrease -dP/dt
max
) were monitored continuously,
recorded, and analyzed after 10 min of stabilization.
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Detection of CK and LDH Activities
The sera of rats were separated by centrifugation, and the
levels of diagnostic marker enzymes CK and LDH in
serum were determined using standard commercial kits
(JianCheng, Nanjing, China).
Detection of SOD, Glutathione (GSH)
Activities, and MDA Concentration
The sera were separated by centrifugation. The SOD activ-
ity, GSH activity, and MDA concentration were measured
using standard commercial kits (Nanjing Jiancheng
Bioengineering Institute, JianCheng, Nanjing, China)
according to the manufacturers instructions. For the pre-
paration of myocardial tissue, a well-ground 10% homo-
genate of heart tissue was centrifuged at 3000 r/min for
1015 min at low temperature to prepare a supernatant for
the determination of biochemical indicators. The heart
tissue homogenate was used to evaluate SOD and GSH
activities and the concentration of MDA using commercial
kits (Jiancheng, Nanjing, China).
Histological Inspection of Myocardium
Rat hearts were extracted rapidly after sacricing the ani-
mals and xed in 4% paraformaldehyde. The hearts were
then cleared, dehydrated, made transparent, impregnated,
and embedded in parafn. Hematoxylin-eosin (H&E) stain-
ing was performed on 5-μm-thick sections.
Ultrastructural Examination of
Mitochondria
Cardiac tissue was excised, trimmed to 1 mm
3
, and xed
in 2.5% glutaraldehyde in 0.1 M sodium phosphate buffer
at pH 7.4 and 4°C for 3 h. Next, it was osmicated in 1%
osmium tetroxide for 1 h at 4°C and prepared for thin
sectioning. After dehydration with a graded ethanol series,
the thin sections were observed and photographed with a
transmission electron microscope (TEM) at 80 kV (7650
TEM, Hitachi, Tokyo, Japan).
Detection of Intracellular ROS
The uorescence of a dihydroethidium probe (DHE, Cat.
Beyotime Institute of Biotechnology, Shanghai, China)
was used to analyze the ROS generation in cardiac tissues.
The ROS production was labeled with red uorescence,
which was visualized and evaluated using high content.
Detection of Intracellular Calcium
Concentration
The calcium concentration was determined in myocardial
tissue and free cardiomyocytes. Heart tissue samples
were homogenized in a 10% (W/V) buffered solution.
The homogenate was centrifuged (1000 rpm, 10 min),
and the upper clear liquid was used for all downstream
biochemical analyses. The calcium level was assessed by
Coomassie Brilliant Blue staining with a commercial kit
(Jian Cheng, Nanjing, China).
Cardiomyocytes were isolated as described below.
Experiments were performed to measure the uorescence
of Cardiomyocytes (CAF-100, Japan Spectroscopic,
Tokyo, Japan). At the beginning of the experiments, cells
in suspension were equilibrated for 23 min before data
collection and incubated with 2 μM fura-2/AM for 30 min
at 37°C. The excitation wavelength alternated between 340
and 380 nm at intervals of 1 s with the emission wave-
length set at 510 nm. The plateau levels of the intracellular
free calcium concentration were used for analysis. The
intracellular free calcium concentration was calculated
using a described previously equation.
19
Isolation of Cardiomyocytes
To obtain normal cardiomyocytes, single ventricular
myocardial cells were isolated from animals that had
been tranquilized via intraperitoneal injections of
heparin sodium (500 IU/kg) and sodium pentobarbital
(40 mg/kg). The heart was quickly cut out of the animal
and canulated on a Langendorff instrument for perfusion
with an oxygenated frostbound free calcium Tyrodes
solution. The solution contained 5.4 mM KCl, 135
mM NaCl, 0.33 mM NaH
2
PO
4
,1.0mMMgCl
2
,10
mM glucose, and 10 mM HEPES. The pH was adjusted
to 7.4 using 3 M NaOH. Next, the heart was perfused
with an enzymatic solution containing Ca
2+
-free
Tyrodes solution with 30 μmol/L of CaCl
2
,0.6mg/
mL of collagenase type II, and 0.5 mg/mL of bovine
serum albumin. After perfusion, the cardiac tissue was
decomposed into fragments in Krebs buffer solution.
The cardiomyocytes were kept in Krebs buffer solution
for up to 1 h before subsequent experiments.
To obtain ischemic cardiomyocytes, ISO (85 mg/kg)
was subcutaneously injected into rats to induce myocardial
ischemia. The dosages and paradigms of injections were
performed according to previous studies.
20
After induction
of myocardial ischemia for two consecutive days, the heart
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was removed and used for the same experiments as the
normal rat ventricular myocytes.
Electrical Recordings of L-Type Ca
2+
Currents
LTCC expression on ventricular myocytes was measured
by the whole cell patch clamp technique. A pipette
puller (Sutter Instruments, Novato, California, USA)
was used to pull borosilicate glass electrodes. I
Ca-L
wasrecordedusinganAxonPatch200Bamplier, and
pCLAMP10.0 software was used for analysis (Axon
Instruments, Union City, CA, USA). The currents were
screened at 2 kHz.
Statistical Analysis
The data obtained were expressed as the mean ± standard
error of the mean (SEM) and were analyzed by one-way
analysis of variance (ANOVA), followed by a Students
t-test using Origin Pro 9.1 software. P < 0.05 was consid-
ered as statistically signicant.
Results
Actions of MAG on Electrocardiography
and Cardiac Functional Parameters
Compared with the Con group (Figure 2), the heart rate
and J-point elevation were dramatically elevated in ani-
mals with ISO-induced MII (p< 0.05 or p< 0.01). This
shows that the MII model was successfully established. A
marked decrease in these two indicators (p< 0.05 or p<
0.01) occurred in the MAG group in comparison to the
ISO group.
Heart rate, LVEDP, and ±dp/dt
max
in ISO-treated
animals were compared to those in the control group.
Tab le 1 shows that the pretreatment of rats with MAG
Figure 2 Actions of MAG on ECG. Representative ECG tracings of each group (A). Statistical analysis of J-point elevation (B) and heart rate (C) in each group. The values
are the mean ± standard deviation (n=10). Compared to the Con group (**p<0.01); Compared to the ISO group (
#
p<0.05); Compared to the ISO group (
##
p<0.01).
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and Ver caused restoration of both parameters to nearly
normal levels. Before the injection of ISO, the groups
pretreated with MAG and Ver showed no signicant
difference from the control and ISO group (p<0.05
or p< 0.01).
Effects of MAG on CK and LDH Activities
MII was appraised by detecting CK and LDH activities
(Figure 3). An obvious elevation was measured in the
activities of CK and LDH in the ISO group in comparison
with the Con group (p< 0.01). Nevertheless, there was an
obvious reduction in the MAG and Ver groups in compar-
ison with the ISO group (p< 0.05 or p< 0.01).
Effects of MAG on SOD, GSH Activities,
and MDA Levels
The oxidative stress response was evaluated by detecting
the activity of SOD, GSH, and MDA levels (Figures 4 and
5). In the serum experiment, the ISO group had obviously
higher MDA levels than the Con group, but SOD and GSH
activity decreased (p< 0.01), as shown in Figure 4. The
MDA levels decreased in the MAG and Ver groups after
treatment, while the SOD and GSH activities increased
(p< 0.01, p< 0.05).
In experiments measuring oxidative stress in myo-
cardial tissue, the ISO group had obviously increased
MDA levels compared to the Con group, but SOD and
GSH activity decreased (p< 0.01), as shown in
Figure 5. The MDA levels decreased in the MAG and
Ver groups, but the SOD and GSH activities increased
(p< 0.01, p< 0.05).
Actions of MAG on Histopathology
Figure 6ADshow the histopathology changes in the rat
hearts detected by light microscopy. Natural muscle bril
structure was detected in the cardiac organization slices
in the Con group (Figure 6A), whereas the ISO group
demonstrated cardiomyocyte swelling, vestigial inltrat-
ing inammatory cells, and the disappearance of trans-
verse striations (Figure 6B). The MAG and Ver groups
showed similarly normal structures with clear transverse
fringes, as well as slight edema and a small amount of
inammatory cardiomyocytes in comparison to the ISO
group (Figure 6C and D).
Table 1 Effects of MAG on Changes of Cardiac Functional Parameters
Group LVEDP (mmHg) LVSP (mmHg) +dp/dt
max
(mmHg/s) -dp/dt
max
(mmHg/s)
Con 5.4 ± 0.68 151.9 ± 17.11 6143.7 ± 761.91 5611.1 ± 710.48
ISO 20.5 ± 1.81** 107.1 ± 14.05** 3743.6 ± 451.29** 3343.8 ± 420.29**
MAG 14.9 ± 1.67
#
126.5 ± 16.34
#
5230.1 ± 549.71
##
4930.6 ± 590.71
##
Ver 9.7 ± 0.76
##
139.2 ± 18.62
#
5822.7 ± 690.06
##
5122.8 ± 621.37
##
Notes: Values are means ± SEM (n=10). **p<0.01 vs Con group.
#
p<0.05 vs ISO group;
##
p<0.01 vs ISO group.
Figure 3 Actions of MAG on cardiac markers. The activities of CK (A) and LDH (B) were determined. The values are the mean ± standard deviation (n=10). Compared to
the Con group (**p<0.01); Compared to the ISO group (
#
p<0.05); Compared to the ISO group (
##
p<0.01).
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Actions of MAG on the Myocardial
Mitochondrion Ultrastructure
As shown in Figure 6, the mitochondrial ultrastructures
were observed by TEM. Cardiomyocytes of the control
animals exhibited regularly shaped cylinders composed of
Z lines with dyads, sarcomeres, elliptical nuclei, numerous
mitochondria, and prominent myolaments (Figure 6E).
Compared to the Con group, the ISO group showed dis-
tinctive ultrastructure alterations. Edema was evident, and
sarcomeres and myolament arrangements were disordered
and partially separated. The mitochondria were enlarged
and rounded, and the cristae appeared disordered or dis-
rupted (Figure 6F). However, the damage to myocardial
ultrastructures in the MAG-treated and Ver-treated groups
was attenuated compared with that of the ISO group
(Figure 6G and H).
Effects of MAG on ROS Release
Figure 7A shows the ROS of rat heart tissue according to
the DHE uorescent probe analysis. MAG and Ver
obviously reduced the production of ROS in cardiomyo-
cytes. As shown in Figure 7A, the intracellular ROS levels
were higher in the ISO group than the Con group
(p< 0.01). Figure 7B shows that the production of ROS
in the ISO group was obviously increased compared to the
Con group, and MAG and Ver evidently reduced the
production of ROS in cardiomyocytes (p< 0.01).
Figure 4 Actions of MAG on the activities of SOD (A), GSH (B), and MDA (C) levels in serum. The values are the mean ± standard deviation (n=10). Compared to the Con
group (**p< 0.01); Compared to the ISO group (
#
p<0.05); Compared to the ISO group (
##
p<0.01).
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Effects of MAG on Calcium Concentration
As shown in Figure 8A, calcium concentrations in the myo-
cardial tissue of the ISO group increased signicantly
(p< 0.01) compared to the Con group, but they signicantly
decreased in the MAG group (p< 0.01) and Ver group
(p< 0.01) in comparison to the ISO group. In the case of
free cardiomyocytes, the ISO group had obviously increased
calcium concentrations (p< 0.01) compared with the Con
group, as shown in Figure 8B. However, the MAG group (p
< 0.01) and Ver group (p< 0.01) showed signicant decreases.
Determination of I
Ca-L
The I
Ca-L
was determined by the steady-state activation
protocol. The application of Ver (0.1 mM), a specic
L-type Ca
2+
channel antagonist, nearly entirely blocked
the currents (Figure 9)(p< 0.01). This indicates that
these currents were L-type Ca
2+
currents.
Inhibitory Effects of MAG on I
Ca-L
Healthy cardiomyocytes and ischemic cardiomyocytes were
exposedto1×10
4
M MAG, and the height of the I
Ca-L
peak
decreased in both cases (p< 0.01). I
Ca-L
remained stable after
washing with an external solution. Figure 10A,Cand Eshow
that MAG can inhibit I
Ca-L
in healthy cardiomyocytes.
Figure 10B,Dand Fshow that MAG can inhibit I
Ca-L
in
ischemic cardiomyocytes and that this inhibition is
irreversible.
Figure 5 Actions of MAG on the activities of SOD (A), GSH (B), and MDA (C) levels in heart tissue. The values are the mean ± standard deviation (n=10). Compared to
the Con group (**p< 0.01); Compared to the ISO group (
##
p<0.01).
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Dose Dependence of MAG on I
Ca-L
Figure 11 demonstrates the current traces obtained by
depolarization with test potentials of 80 to 0 mV at
various MAG concentrations. I
Ca-L
was gradually inhibited
by raising concentrations of MAG from 10
6
to 10
4
M.
The peak amplitudes of I
Ca-L
were reduced by 11.46 ±
0.80%, 18.76 ± 0.81%, 29.19 ± 2.10%, 45.33 ± 0.63%, and
54.32 ± 2.53%. MAG progressively suppressed I
Ca-L
in a
dose-dependent manner. The semi-maximal prohibitive
concentration of MAG was calculated as 14 μM.
Actions of MAG on the Current-Voltage
Relationship of I
Ca-L
Figure 12 shows the effects of MAG (10
6
, 10,
5
10
4
M)
on the current-voltage relationship of I
Ca-L
.Figure 12A
shows cross-sectional traces with sequential treatments of
10
6
,10
5
and 10
4
M of MAG. Figure 12B exhibits the
current-density-voltage relationship with controls for 10
6
,
10
5
and 10
4
M of MAG and 10 μM of Ver. These results
indicate that the different MAG concentrations signi-
cantly decreased the maximum current.
Figure 7 Actions of MAG on reactive oxygen species in rat heart tissue by uorescent probe DHE. Representative sections are from the heart of the Con (A
1
), ISO (A
2
),
MAG (A
3
), and Ver (A
4
) groups. Representative sections are from semi-quantitative calculation of ROS content in rat myocardium (B). The values are the mean ± standard
deviation. Compared to the Con group (**p<0.01); Compared to the ISO group (
##
p<0.01). Scale bar: 50 μm.
Figure 6 Actions of MAG on histopathological changes of animalscardiac tissue colored with H&E and ultrastructural changes. Representative sections (magnication:
400×) were from the heart of the Con (A), ISO (B), MAG (C), and Ver (D) groups. Scale bar: 50 μm. The ultrastructure of heart tissues was detected by Transmission
Electron Microscope (magnication: 15,000×) from the left ventricle of the Con (E), ISO (F), MAG (G), and Ver (H) groups. Scale bar: 1.0 μm.
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Steady Activation and Inactivation of Ca
2+
Current by MAG
Figure 13 presents the dependence of the steady-state activa-
tion and inactivation of Ca
2+
current in the presence and
absence of MAG (10
6
,10,
5
and 10
4
M). The V
1/2
value
of the normalized activation conductivity curve was 10.59 ±
0.74 mV, and the slope (k) of the control group was 7.67 ±
0.68 mV. The other V
1/2
values were 12.95 ± 0.59 mV (k =
7.27 ± 0.53 mV) for 10
6
mol/L of MAG, 12.37 ± 0.44 mV
(k = 7.43 ± 0.40 mV) for 10
5
mol/L MAG, and 14.18 ±
0.51 mV (k = 7.12 ± 0.46 mV) for 10
4
mol/L of MAG.
The V
1/2
values of the steady-state inactivation were
30.55 ± 0.74 mV (k = 5.16 ± 0.72 mV) for the control,
33.25 ± 0.45 mV (k = 5.11 ± 0.40 mV) for 10
6
mol/L of
MAG, 34.38 ± 0.40 mV (k = 5.19 ± 0.34 mV) for 10
5
mol/L of MAG, and 35.25 ± 0.23 mV (k = 5.27 ± 0.20
mV) for 10
4
mol/L of MAG. The results indicate that
MAG did not change the activation and inactivation of
gated characteristics of LTCC (p> 0.05). With or without
MAG, there was no signicant difference in the normal
activation and inactivation values of V
1/2
(p> 0.05).
Discussion
IHD is a major type of cardiovascular disease, particularly
myocardial infarction. The cellular mechanisms underly-
ing the ISO-induced MII or hypoxia in the MII model
Figure 8 Actions of MAG on Ca
2+
concentrations levels. (A) Calcium content in myocardial tissue homogenate. (B)IntracellularCa
2+
concentration in isolated ventricular myocytes
with uorescence spectrophotometer. The values are the mean ± standard deviation (n=10). Compared to the Con group (**p<0.01); Compared to the ISO group (
##
p<0.01).
Figure 9 Ver (0.1 mM) wholly suppressed the Ca
2+
current in cardiomyocytes. (A) Typical traces under verapamil treatment. (B) Summary data of A. The values are the
mean±standard deviation (n=10 cells). **p<0.01 compared with Con.
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Figure 10 Actions of MAG on I
Ca-L
of healthy cardiomyocytes (A,C,E) and ischemic cardiomyocytes (B,D,F). MAG I
Ca-L
under control conditions, 1×10
4
M MAG, and
washout. The values are the mean ± standard deviation (n=10 cells). **p<0.01 compared with Con.
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includes cardiac muscle hyperactivity and intracellular
calcium ion inux. Furthermore, the pathophysiology
changes in the ISO-based MII model resemble the results
observed in humans.
21,22
Licorice is one of the oldest medicinal plants and con-
tains 300 active ingredients. Licorice and its extracts have
antibacterial, antiviral, anti-inammatory, anticancer, anti-
oxidant, and other activities. MAG is one of the important
effective ingredients and has good effects in the treatment of
cardiovascular disease.
23,24
It is widely used in clinical
immunoregulation, the promotion of bilirubin metabolism,
the suppression viral hepatitis, improvement of liver func-
tion, adjuvant therapy for tumor chemoradiotherapy, and
polycystic ovary syndrome, among others.
23,24
In this study,
we investigated the cardioprotective effects and possible
mechanisms of MAG injection on ISO-induced MII.
ISO is a β-receptor agonist, and continuous high doses
can induce myocardial infarction-like injury in experimental
animals. ISO is often used in models of myocardial
ischemia.
25
The literature suggests that the key factors of
ischemic injury are the decrease of Ca
2+
uptake in the sarco-
plasmic reticulum of the ischemic myocardium and intracel-
lular calcium ion inux. The myocardium is extremely
sensitive to hypoxia. Hypoxia can cause intracellular calcium
ion inux, which is an important mechanism that leads to
myocardial cell injury and necrosis. After the intervention of
Figure 11 Actions of MAG at distinct concentrations on I
Ca-L
.(A) Current traces of I
Ca-L
upon immerse to 1×10
6
, 3×10
6
, 1×10
5
, 3×10
5
, and 1×10
4
M MAG, as well as
0.1 mM Ver. (B) Time course of I
Ca-L
upon immersion in 1×10
6
, 3×10
6
, 1×10
5
, 3×10
5
, and 1×10
4
M MAG, as well as 0.1 mM Ver. (C) Concentration-response curve of
MAG. The values are the mean ± standard deviation (n=10 cells).
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MAG and Ver, the concentration of free calcium in cardio-
myocytes decreased signicantly (Figure 8A and B). This
shows that MAG has a similar effect to that of a calcium
antagonist.
Systolic and diastolic dysfunction and altered cardiac
structure are some of the characteristics of an ISO-induced
myocardial ischemia model. In this study, we evaluated the
hemodynamic and histological changes, the decrease of
LVSP and ±dp/dt
max
, the increase of LVEDP (Table 1),
and the injury of myocardial tissue to conrm the effect of
the ISO-induced myocardial ischemia model in rats. Under
a light microscope, H&E staining sections showed that the
Figure 12 Actions of MAG on the I-V relationship of I
Ca-L
. Example tracks (A) and assembled information (B) were obtained with the following treatments: Con (),1×10
6
MMAG(), 1×10
5
MMAG(), 1×10
4
MMAG(), and 0.1 mM Ver (). The values are the mean ± standard deviation (n=10 cells).
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myocardial tissue of the ISO group was obviously
damaged, the arrangement of myocardial cells was disor-
dered, the muscle space was widened, the nucleus was
pyknotic, there was deformation or dissolution, and the
membrane was damaged (Figure 6AD).
The ultrastructures of the myocardium were also
observed under a light microscope. In the ISO group,
some mitochondria were swollen, fused, or cristaed, and
the Z-line became thinner. The MAG group and Ver group
showed signicantly improvements in these morphological
changes (Figure 6EH). Therefore, treatment with MAG
and Ver can improve heart function.
CK and LDH are cytoplasmic enzymes that are
released only in the vent of myocardial cell damage,
which increases their activity. Therefore, we used CK
and LDH as biochemical markers to detect cardiac cellular
changes under MII conditions induced by ISO, as well as
the intracellular calcium concentration. The results are
shown in Figure 3. When oxidative stress is stimulated,
the balance between pro-oxidation and anti-oxidation in
the body tends toward pro-oxidation, which leads to home-
ostasis disorder along with tissue and cell damage.
Oxidative stress is also one of the important reasons for
the abnormal structure and function of the cardiovascular
system. Oxidative stress plays a key role in the occurrence
and development of cardiovascular diseases, such as ather-
osclerosis, myocardial ischemia-reperfusion damage, and
arrhythmia.
There are many kinds of free radicals, and ROS are
closely related to oxidative stress. Cells metabolize oxi-
dants, and they have evolved enzymatic or non-enzymatic
mechanisms to resist oxidant toxicity, such as myocardial
and serum SOD, GSH, and MDA.
26
Often, a small amount
of free radicals is produced in normal tissue metabolism,
and the balance of oxygen free radicals is maintained
through intracellular defense mechanisms. However,
under the action of some damage factors, the oxidative
metabolites in cells increase, or the antioxidant protection
mechanism becomes insufcient. As a result, free radicals
accumulate and produce toxic effects on cells.
This imbalance between oxidation and antioxidants is
called oxidative stress. Oxidative stress directly induces the
denaturation of cytosolic proteins and enzymes, apoptosis or
cell death, tissue damage, and disease.
27
The scavenging
mechanism of ROS includes primary and secondary antiox-
idant defense systems. Myocardial and serum SOD, GSH
Figure 13 Actions of MAG on steady-state activation and inactivation of Ca
2+
current. Activation dynamics (A) and inactivation dynamics of Ca
2+
current (B) are
demonstrated for the following treatments: Con (), 1×10
4
MMAG(), 1×10
5
MMAG(), and MAG at 1×10
6
MMAG(). The values are the mean ± standard
deviation (n=10 cells).
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activity, MDA content, and intracellular ROS were detected
in experiments, and the results are shown in Figures 4,5,
and 7.
We tested the effects of MAG on myocardial cells and
found that it reduced the calcium concentration in cardiac
tissue.
28
Therefore, we used the whole-cell patch-clamp
technique to investigate the effects of MAG on the intra-
cellular conditions and ion channels. The patch-clamp
results illustrate that MAG decreased I
Ca-L
in a concentra-
tion-dependent fashion, and the semi-maximal prohibitive
concentration was 14 μM, as shown in Figure 11. This
might have resulted from the MAG combining with the
channel proteins by forming covalent bonds.
29
The I-V relationship or I
Ca-L
reverse potential remained
unchanged (Figure 12). MAG inhibited the peak value of
the Ca
2+
current without affecting the steady-state activa-
tion and inactivation of I
Ca-L
(Figure 13). The physiological
process of converting an electrical stimulus to a mechanical
response was characterized by excitation-contraction (EC)
coupling.
30
In EC coupling, voltage-gated calcium channels
are activated by membrane depolarization, and then Ca
2+
enters the myocytes through LTCCs. Ca
2+
entry through the
nearby LTCCs then triggers the release of Ca
2+
from rya-
nodine receptors.
31
The decrease of calcium results in less
of a decrease in myocardial oxygen consumption, which
reduces the calcium concentration in cardiac myocytes and
can alleviate arrhythmia and cell damage. We found that
MAG had signicant but conditional inhibitory effects on
I
Ca-L
.
The data obtained from organs, tissues, and cells in
vitro can be used to infer about the role of MAG in vivo,
but our experiments in isolated cells were carried out at
room temperature (25 degrees celsius). One of the limita-
tions of this study is that there is a gap between this
experimental environment and the physiological environ-
ment of cardiomyocytes. Therefore, the direct effect of
MAG on I
Ca-L
needs further study.
Conclusion
We demonstrated the protective effects of MAG injection
in an ISO-induced MII model. The mechanism underlying
the cardioprotective effect might be related to the regula-
tion of oxidative stress and the reduction of calcium inux
by inhibiting LTCCs. These results could provide further
insight into the molecular mechanisms underlying the ben-
ecial effects of MAG injection on IHD.
Acknowledgments
This work was supported by Research Foundation of
Hebei University of Chinese Medicine (No. KTZ
2019041). Zhifeng Zhao and Miaomiao Liu are co-rst
authors for this study.
Disclosure
The authors declare no conicts of interest in this work.
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... [27][28][29] Previous studies have reported that MgIG has strong myocardial protections, including anti-myocardial fibrosis, antimyocardial ischemia (MI), and infarction. 30,31 Among them, Ma et al demonstrated that MgIG could reduce myocardial fibrosis in mice induced by isoproterenol (ISO), and the protective effects might be relevant to the decreased expression levels of NF-κB. 30 Zhao et al have demonstrated that monoammonium glycyrrhizinate, an aglycone of glycyrrhizin that can be extracted from liquorice, provides protection against ISO-induced MI damage (in vivo and in vitro). ...
... 30 Zhao et al have demonstrated that monoammonium glycyrrhizinate, an aglycone of glycyrrhizin that can be extracted from liquorice, provides protection against ISO-induced MI damage (in vivo and in vitro). 31 Furthermore, we found that MgIG can reduce the concentration of intracellular calcium ions and the contractility of myocardial cells by inhibiting the L-type calcium current of myocardial cells, which has a similar effect to calcium antagonists. 32 However, to our knowledge, no study has explored the protection offered by MgIG in ATO-induced cardiotoxicity or the underlying mechanism. ...
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... Reestablishing this Ca 2+ homeostasis has a cardioprotective effect against MI injury. 12 Augmented oxidative stress is one of the major elements of the complex pathophysiological mechanism underlying MI. 13 Oxidative stress results from the increased formation of reactive oxygen species (ROS) and causes DNA damage, protein and lipid peroxidation, and cellular dysfunction, all of which are associated with the pathological injury of the heart. 14 In most cell types, mitochondria are the primary source of ROS, 15 yet the damage ROS cause is not restricted to mitochondrial macromolecules but extends to the surrounding intracellular space as well. ...
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... Liquiritin has a variety of biological effects, such as neuroprotective effect, enhancing the ability of cells to resist oxidative stress, alleviating mitochondrial damage, regulating mitochondrial quality control, and alleviating myocardial pathological structural changes in mice with myocardial fibrosis [38,39]. Zhao et al. found that monoammonium glycyrrhizinate can improve cardiac morphology, repair mitochondrial damage, inhibit oxidative stress and reduce the production of reactive oxygen species by testing SOD and GSH activities, MDA concentration, histological myocardium inspection, mitochondria ultrastructure changes [40]. Based on the above studies on the pharmacological effects of various components in QJJLD, we believe that they play a synergistic role in improving gastrocnemius atrophy in EAMG rats by restoring mitochondrial biogenesis and antioxidant stress. ...
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... Mitochondria injury was further validated with mitochondria oxidative stress marker SOD and MDA, and membrane damage indicator LDH in transfected A549 cells. 21,22 As expected, activity of SOD, MDA and LDH exhibited the same trend with JC-1 labeled flow cytometry ( Figure 4B, *p<0.05 versus control, and # p<0.05 versus miR-199a-3p or pcDNA-ZEB1). ...
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Background: Most cultured enzymatically dissociated adult myofibers exhibit spatially uniform (UNI) contractile responses and Ca(2+) transients over the entire myofiber in response to electric field stimuli of either polarity applied via bipolar electrodes. However, some myofibers only exhibit contraction and Ca(2+) transients at alternating (ALT) ends in response to alternating polarity field stimulation. Here, we present for the first time the methodology for identification of ALT myofibers in primary cultures and isolated muscles, as well as a study of their electrophysiological properties. Results: We used high-speed confocal microscopic Ca(2+) imaging, electric field stimulation, microelectrode recordings, immunostaining, and confocal microscopy to characterize the properties of action potential-induced Ca(2+) transients, contractility, resting membrane potential, and staining of T-tubule voltage-gated Na(+) channel distribution applied to cultured adult myofibers. Here, we show for the first time, with high temporal and spatial resolution, that normal control myofibers with UNI responses can be converted to ALT response myofibers by TTX addition or by removal of Na(+) from the bathing medium, with reappearance of the UNI response on return of Na(+). Our results suggest disrupted excitability as the cause of ALT behavior and indicate that the ALT response is due to local depolarization-induced Ca(2+) release, whereas the UNI response is triggered by action potential propagation over the entire myofiber. Consistent with this interpretation, local depolarizing monopolar stimuli give uniform (propagated) responses in UNI myofibers, but only local responses at the electrode in ALT myofibers. The ALT responses in electrically inexcitable myofibers are consistent with expectations of current spread between bipolar stimulating electrodes, entering (hyperpolarizing) one end of a myofiber and leaving (depolarizing) the other end of the myofiber. ALT responses were also detected in some myofibers within intact isolated whole muscles from wild-type and MDX mice, demonstrating that ALT responses can be present before enzymatic dissociation. Conclusions: We suggest that checking for ALT myofiber responsiveness by looking at the end of a myofiber during alternating polarity stimuli provides a test for compromised excitability of myofibers, and could be used to identify inexcitable, damaged or diseased myofibers by ALT behavior in healthy and diseased muscle.
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Increased reactive oxygen species (ROS) production and elevated intracellular Ca2+ following cardiac ischemia-reperfusion injury are key mediators of cell death and the development of cardiac hypertrophy. The L-type Ca2+ channel is the main route for calcium influx in cardiac myocytes. Activation of the L-type Ca2+channel leads to a further increase in mitochondrial ROS production and metabolism. We have previously shown that the application of a peptide derived against the alpha-interacting domain of the L-type Ca2+ channel (AID) decreases myocardial injury post reperfusion. Herein, we examine the efficacy of simultaneous delivery of the AID peptide in combination with the potent antioxidants curcumin and resveratrol using multifunctional poly(glycidyl methacrylate) (PGMA) nanoparticles. We highlight that drug loading and dissolution are important parameters that have to be taken into account when designing novel combinatorial therapies following cardiac ischemia-reperfusion injury. In the case of resveratrol low loading capacity and fast release rates hinders its applicability as an effective candidate for simultaneous therapy. However in the case of curcumin, high loading capacity and sustained release rates enables its effective simultaneous delivery in combination with the AID peptide. Simultaneous delivery of both compounds allowed for effective attenuation of L-type Ca2+ channel-activated increases in superoxide ( assessed as changes in DHE fluorescence; Empty NP = 53.1±7.6%; NP-C-AID = 7.32±3.57%) and mitochondrial membrane potential (assessed as changes in JC-1 fluorescence; Empty NP = 19.8±2.8%; NP-C-AID=13.05±1.78%). We demonstrate in isolated rat hearts exposed to ischemia followed by reperfusion, that curcumin and the AID peptide in combination effectively reduce muscle damage, decrease oxidative stress and superoxide production in cardiac myocytes.
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Entecavir (ETV) is a superior nucleoside analogue used to treat hepatitis B virus (HBV) infection. Although its advantages over other agents include low viral resistance and the elicitation of a sharp decrease in HBV DNA, adverse effects such as hepatic steatosis, hepatic damage and lactic acidosis have also been reported. Glycyrrhizin has long been used as hepato-protective medicine. The clinical combination of ETV plus glycyrrhizin in China displays better therapeutic effects and lower rates of liver damage. However, there is little evidence explaining the probable synergistic mechanism that exists between these two drugs from a pharmacokinetics view. Here, alterations in the plasma pharmacokinetics, tissue distribution, subcellular distribution, and in vitro and in vivo antiviral activity of ETV after combination with glycyrrhizic acid (GL) were analysed to determine the synergistic mechanisms of these two drugs. Specific efflux transporter membrane vesicles were also used to elucidate their interactions. The primary active GL metabolite, glycyrrhetic acid (GA), did not affect the plasma pharmacokinetics of ETV but promoted its accumulation in hepatocytes, increasing its distribution in the cytoplasm and nucleus and augmenting the antiviral efficiency of ETV. These synergistic actions were primarily due to the inhibitory effect of GA on MRP4 and BCRP, which transport ETV out of hepatocytes. In conclusion, GA interacted with ETV at cellular and subcellular levels in the liver through MRP4 and BCRP inhibition, which enhanced the antiviral activity of ETV. Our results partially explain the synergistic mechanism of ETV and GL from a pharmacokinetics view, providing more data to support the use of these compounds together in clinical HBV treatment.
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Context: Drug-induced liver injury (DILI) is associated with altering expression of hepatobiliary membrane transporters. Monoammonium glycyrrhizin (MAG) is commonly used for hepatic protection and may have a correlation with the inhibition effect of multidrug resistance-associated protein 2 (Mrp2). Objective: This study evaluates the dynamic protective effect of MAG on rifampicin (RIF)- and isoniazid (INH)-induced hepatotoxicity in rats. Materials and methods: Male Wistar rats were randomly divided into four groups of 15 rats. Liver injury was induced by co-treatment with RIF (60 mg/kg) and INH (60 mg/kg) by gavage administration; MAG was orally pretreated at the doses of 45 or 90 mg/kg 3 h before RIF and INH. Rats in each group were sacrificed at 7, 14, and 21 d time points after drug administration. Results: Liver function, histopathological analysis, and oxidative stress factors were significantly altered in each group. The expression of Mrp2 was significantly increased 230, 760, and 990% at 7, 14, and 21 time points, respectively, in RIF- and INH-treated rats. Compared with the RIF and INH groups, Mrp2 was reduced and Ntcp was significantly elevated by 180, 140, and 160% in the MAG high-dose group at the three time points, respectively. The immunoreaction intensity of Oatp1a4 was increased 170, 190, and 370% in the MAG low-dose group and 160, 290, and 420% in the MAG high-dose group at the three time points, respectively, compared with the RIF and INH groups. Discussion and conclusion: These results indicated that MAG has a protective effects against RIF- and INH-induced hepatotoxicity. The underlying mechanism may have correlation with its effect on regulating the expression of hepatobiliary membrane transporters.
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Aims: Ischemic heart disease is a leading cause of death and disability worldwide. Despite recent advances, there is no effective therapy for preventing myocardial ischemia-reperfusion (I/R) injury. In this study, we aimed to examine the therapeutic effect of scutellarin, a flavone isolated from the traditional Chinese medicine Scutellaria barbata and Erigeron breviscapus, on cardiomyocyte I/R injury. Main methods: Neonatal rat cardiomyoblast cells H9C2 were used to study the role of scutellarin in cardiomyocyte injury. I/R injury was induced by 2h of hypoxia plus glucose and serum deprivation, followed by 6-hour recovery. Cardiomyocyte damage was evaluated by the release of pro-inflammatory cytokines and creatine kinase (CK), apoptosis, and cell proliferation. Oxidative responses were assessed by reactive oxygen species (ROS) production, MDA generation, SOD expression, and mitochondrial membrane potential detection. Activation of JAK2/STAT3 signaling and expression of pro- or anti-survival molecules were detected by Western blot. Key findings: I/R injury increased the release of CK as well as pro-inflammatory cytokines TNFα, IL-1β, IL-6, and IL-8 from cardiomyocytes. ROS, MDA, and apoptosis were enhanced in cardiomyocytes underwent I/R injury, while cell proliferation, mitochondrial membrane potential, SOD expression were reduced. Scutellarin treatment dose-dependently suppressed I/R injury-induced pro-inflammatory cytokine and CK release, oxidative response, loss of mitochondrial membrane potential, and enhanced cell proliferation and anti-oxidant SOD expression. Further analysis suggests scutellarin promotes JAK/STAT3 activation and expression of pro-survival proteins Bcl2, VEGF, MMP2, and MMP9. Pro-apoptotic molecules Bax and caspase-3 were suppressed by scutellarin. Significance: We identified a previously unrecognized pathway by which scutellarin protects myocardial I/R injury. Scutellarin modulates I/R injury-induced oxidative stress and apoptosis probably by enhancing JAK2/STAT3 pro-survival signaling.