Chin J Integr Med 2022 Nov;28(11):983-991 • 983 •

C
Available online at link.springer.com/journal/11655
hinese Journal of Integrative Medicine Journal homepage: www.cjim.cn/zxyjhen/zxyjhen/ch/index.aspx
E-mail: cjim_en@cjim.cn

Original Article
Shenmai Injection Attenuates Myocardial Ischemia/Reperfusion
Injury by Targeting Nrf2/GPX4 Signalling-Mediated Ferroptosis
MEI Sheng-lan, XIA Zhong-yuan, QIU Zhen, JIA Yi-fan, ZHOU Jin-jian, and ZHOU Bin

ABSTRACT Objective:
Objective : To examine the effect of Shenmai Injection (SMJ) on ferroptosis during
myocardial ischemia reperfusion (I/R) injury in rats and the underlying mechanism. Methods Methods:: A total of 120
SPF-grade adult male SD rats, weighing 220–250 g were randomly divided into different groups according
to a random number table. Myocardial I/R model was established by occluding the left anterior descending
artery for 30 min followed by 120 min of reperfusion. SMJ was injected intraperitoneally at the onset of
120 min of reperfusion, and erastin (an agonist of ferroptosis), ferrostatin-1 (Fer-1, an inhibitor of ferroptosis)
and ML385 (an inhibitor of nuclear factor erythroid-2 related factor 2 (Nrf2)) were administered intraperitoneally
separately 30 min before myocardial ischemia as different pretreatments. Cardiac function before ischemia, after
ischemia and after reperfusion was analysed. Pathological changes in the myocardium and the ultrastructure of
cardiomyocytes were observed, and the myocardial infarction area was measured. Additionally, the concentration
of Fe2+ in heart tissues and the levels of creatine kinase-MB (CK-MB), troponin I (cTnI), malondialdehyde
(MDA) and superoxide dismutase (SOD) in serum were measured using assay kits, and the expressions of
Nrf2, glutathione peroxidase 4 (GPX4) and acyl-CoA synthetase long-chain family member 4 (ACSL4) were
examined by Western blot. Results
Results:: Compared with the sham group, I/R significantly injured heart tissues, as
evidenced by the disordered, ruptured and oedematous myocardial fibres; the increases in infarct size, serum
CK-MB, cTnI and MDA levels, and myocardial Fe2+ concentrations; and the decreases in SOD activity (P <0.05).
These results were accompanied by ultrastructural alterations to the mitochondria, increased expression of
ACSL4 and inhibited the activation of Nrf2/GPX4 signalling (P <0.05). Compared with the I/R group, pretreatment
with 9 mL/kg SMJ and 2 mg/kg Fer-1 significantly reduced myocardial I/R injury, Fe2+ concentrations and
ACSL4 expression and attenuated mitochondrial impairment, while 14 mg/kg erastin exacerbated myocardial
I/R injury (P <0.05). In addition, cardioprotection provided by 9 mL/kg SMJ was completely reversed by ML385,
as evidenced by the increased myocardial infarct size, CK-MB, cTnI, MDA and Fe2+ concentrations, and the
decreased SOD activity (P <0.05). Conclusions
Conclusions:: Ferroptosis is involved in myocardial I/R injury. Pretreatment with
SMJ alleviated myocardial I/R injury by activating Nrf2/GPX4 signalling-mediated ferroptosis, thereby providing a
strategy for the prevention and treatment of ischemic heart diseases.
KEYWORDS ferroptosis, Shenmai Injection, myocardial ischemia reperfusion injury, pretreatment

Ischemic heart disease is currently one of the
leading causes of death worldwide, and it typically
occurs in younger individuals, which seriously affects
quality of life and increases social burdens.(1) Restoring
the blood supply to the ischemic myocardium is the only
effective treatment for ischemic heart disease. However,
when blood flow is restored, damage to cardiomyocyte
ultrastructure, function and energy metabolism will
be further exacerbated and result in arrhythmia and
cardiac dysfunction, which is called myocardial ischemia
reperfusion (I/R) injury. Studying the underlying
molecular mechanisms of myocardial I/R injury and
examining strategies to prevent and treat ischemic heart
disease have become important issues worldwide.(2)
Previous studies have demonstrated that
programmed cell death (PCD) is involved in the
pathophysiology of myocardial I/R injury.(3-5) Ferroptosis,
©The Chinese Journal of Integrated Traditional and Western
Medicine Press and Springer-Verlag GmbH Germany, part of
Springer Nature 2022
Supported by the National Natural Science Foundation of China
(No. 81970722)
Department of Anaesthesiology, Renmin Hospital of Wuhan
University, Wuhan (430060), China
Correspondence to: ZHOU Bin, E-mail: lvtingzhou@163.com
DOI: https://doi.org/10.1007/s11655-022-3620-x
• 984 •
which was first reported by Dixon, et al (6) in 2012,
is a new and unique type of PCD caused by lipid
peroxidation that is dependent on intracellular reactive
oxygen species (ROS) and iron overload. Ferroptosis is
different from other forms of PCD at the morphological,
biological and genetic levels; it is characterized by the
accumulation of ROS and lipid peroxidation during the
Fenton reaction and impairs the structure and function
of mitochondria. Ferroptosis is associated with a variety
of pathologies, including neurodegeneration, ischemic
stroke, liver and renal injury, tumours, and myocardial
I/R injury. (7-12) A recent study demonstrated that
ferroptosis may be a major cause of cardiac dysfunction
and cardiomyocyte death during myocardial I/R injury.(13)
Inhibiting ferroptosis could markedly attenuate
myocardial I/R injury, but the underlying molecular
mechanisms are still unclear.
Nuclear factor erythroid-2 related factor 2 (Nrf2)
is an important transcription factor that regulates
cellular oxidative stress and is also a central regulator
of intracellular redox homeostasis.(14) Studies have
shown that Nrf2-mediated PCD, such as apoptosis
and autophagy, plays an indispensable role in the
pathology of myocardial I/R injury. (15) Glutathione
peroxidase 4 (GPX4) is a membrane lipid repair
enzyme that can degrade certain small molecular
peroxides and lipid peroxides and inhibit lipid
peroxidation.(16) GPX4 is considered a key regulator
of ferroptosis; it resists iron- and oxygen-dependent
lipid peroxidation by converting lipid peroxides into
nontoxic lipids, thus inhibiting ferroptosis.(17) Nrf2 is
involved in cellular iron accumulation, and GPX4 has
been shown to be a downstream factor of Nrf2. Nrf2/
GPX4 signalling has been suggested to play important
roles in the induction, promotion and regulation
of ferroptosis. (18,19) However, whether Nrf2/GPX4-
mediated ferroptosis participates in myocardial I/R
injury needs to be determined.
Shenmai Injection (SMJ, 参麦注射液), an injection
composed of Radix Ginseng , Radix ophiopogonis
and Schisandra chinensis , is a traditional Chinese
medicine that has long been used to prevent and treat
cardiovascular diseases, but the underlying mechanism
is still unclear.(20) The active components of SMJ, such
as ginsenosides Rb1, Rb2, Rc, Rd, Re and Rg1, have
been successfully isolated and shown to have the effects
of scavenging oxygen free radicals and inhibiting lipid
peroxidation.(21) Recent studies have been performed
Chin J Integr Med 2022 Nov;28(11):983-991
in ICR mice and C57BL/6 mice that demonstrated SMJ
could ameliorate doxorubicin-induced cardiotoxicity
by inhibiting inflammation, maintaining mitochondrial
homeostasis and repairing heart dysfunction. (22,23)
In addition, SMJ has been shown to protect H9C2
cardiomyocytes from hypoxia/reoxygenation injury, which
indicated that pretreatment with SMJ may be a potential
therapeutic strategy for myocardial I/R injury.(23) Previous
studies have reported that SMJ exerts cardioprotective
effects on the myocardium after I/R injury by targeting
Nrf2 signalling, but whether ferroptosis mediated by
Nrf2/GPX4 signalling is involved in this process has not
been determined.
In the present study, we hypothesized that
ferroptosis mediated by Nrf2/GPX4 signalling may be
responsible for the myocardial damage caused by I/R
injury and SMJ could attenuate myocardial I/R
injury by targeting Nrf2/GPX4 signalling and
interrupt ferroptosis. Myocardial I/R rat models were
established to elucidate the relationship between SMJ
and ferroptosis and the role of SMJ in myocardial I/R
injury to provide a new strategy to prevent and treat
ischemic heart diseases.
METHODS
Experimental Animals
A total of 120 SPF-grade male Sprague–Dawley
rats weighing 220–250 g were provided by Beijing
Weitonglihua Experimental Animal Technology Co.,
Ltd., China (SCXL Jing 2016-0006). Experimental
rats were housed in an environment with a controlled
temperature of 24 ℃, a relative humidity of 50%±10%,
and a fixed light/dark schedule (12 h light/12 h dark)
and were allowed free access to food and water.
All experimental procedures were conducted in
accordance with the Guidelines for the Care and Use
of Laboratory Animals of the Chinese Animal Welfare
Committee and the Guidelines of Renmin Hospital of
Wuhan University (No. WDRM 20190414).
Myocardial I/R Injury Model
A well-established myocardial I/R injury model
was used in the present study. (3-5) All rats were
anaesthetized by an intraperitoneal injection of
sodium pentobarbital (50 mg/kg, lot No. 860901,
Sigma-Aldrich, Merck KGaA, USA) and subjected to
tracheotomy and ventilation. The I/R injury model was
established by occluding the left anterior descending
artery for 30 min, followed by 120 min of reperfusion.
Chin J Integr Med 2022 Nov;28(11):983-991
Sham-operated rats were subjected to the same
surgical procedures without ligation. Ischemia was
confirmed by an increase in the ST segment of limb
lead Ⅱ, as well as discolouration of the ischemic
zone. Blood and heart tissue samples were collected
for analysis after reperfusion.
Experimental Protocol
First, the effects of SMJ on myocardial I/R injury
and the optimal dose of SMJ to treat myocardial
damage were examined. Rats were randomly
assigned to 5 groups (n =12): the sham group (sham),
myocardial I/R injury group (I/R), 6 mL/kg SMJ+I/R
(SMJ-L) group, 9 mL/kg SMJ+I/R (SMJ-M) group,
and 12 mL/kg SMJ+I/R (SMJ-H) group. The rats were
numbered, then, according to the random number
table, the number was selected at the beginning of
sampling, finally, sample numbers were drawn. Our
data showed that SMJ-M was the optimal dose to
reduce myocardial I/R injury.(24) Then, the underlying
mechanisms of this cardioprotective effect were
investigated, and the rats were randomly divided
into 5 groups (n =12 in each group): a ferroptosis
inhibitor ferrostatin-1+I/R (Fer-1) group, a ferroptosis
agonist erastin + I/R (Erastin) group, a SMJ + erastin+
I/R (SMJ+Erastin) group, and a SMJ + Nrf2 inhibitor
ML385+ I/R (SMJ+ML385) group and a sham + ML385
group. SMJ (batch No. 010873, Hangzhou Zhengda
Qingchunbao Pharmaceutical Co., Ltd., China) was
injected intraperitoneally at the onset of 120 min of
reperfusion. Fer-1 (lot No. S7243-01, Sellcct Inc., USA)
2 mg/kg dissolved in 1% DMSO,(25) erastin (lot No.
S7242-01, Sellcct Inc., USA) 14 mg/kg dissolved in 1%
DMSO,(26) ML385 (lot No. S8790-01, Sellcct Inc., USA)
30 mg/kg dissolved in 1% DMSO(27) and the vehicle
(0.9% NaCl dissolved in 1% DMSO), which served as
a control, were administered intraperitoneally 30 min
before myocardial ischemia.
Cardiac Function Assessment
Invasive haemodynamic monitoring was performed
to evaluate myocardial function. Heart rate (HR), left
ventricular systolic pressure (LVSP), and maximal rates
of left ventricular-developed pressure increases and
decreases (±dp/dt) were continuously monitored by
electrophysiology (BioPAC, MH150, USA), and the data
were analyzed by AcqKnowledge 8.0 software.
Infarct Size Determination
After heart harvested, the rats were decapitated
• 985 •
in accordance with the Guidelines for the Care and
Use of Laboratory Animals of the Chinese Animal
Welfare Committee. Myocardial infarct size was
measured as described previously.(3,4) Two percent
Evans blue dye and 1% 2,3,5-triphenyltetrazolium
chloride (both from Sigma-Aldrich; Merck KGaA,
USA) were used for myocardial staining. The stained
myocardial slices were scanned and assessed using
an image analysis system (Image-Pro Plus 3.0, Media
Cybernetics, USA). The risk areas were stained red,
while the infarct areas remained pale.
Haematoxylin-Eosin Staining
After 2 h of reperfusion, the heart was
excised, washed with a saline solution, fixed in
4% paraformaldehyde, and embedded in paraffin.
Five-micrometre-thick slices of paraffin-embedded
hearts were sectioned laterally at the level of
left ventricle papillary muscles and stained with
haematoxylin-eosin (HE). The pathological
morphology of the myocardium was observed under a
normal microscope (Olympus DP73, Japan).
Transmission Electron Microscopy
Changes in mitochondria were observed
using transmission electron microscopy. A total of
1 mm3 of the ischemic myocardial border zone was
removed from each heart and then prefixed in a
solution containing 2.5% glutaraldehyde and 1%
osmium tetroxide, postfixed in 1% osmium tetroxide,
dehydrated in an ascending series of alcohols and
embedded in epoxy resin. Then, the stained slides
were photographed under a transmission electron
microscope (Hitachi, HT7700, Japan).
Creatine Kinase MB and Troponin Assay
Blood samples were collected and centrifuged
at 3,000 r/min for 15 min after 120 min of reperfusion,
and the serum was collected to measure creatine
kinase MB (CK-MB, batch No. C060-a) and troponin
(cTnI) levels using commercial kits (Elabscience
Biotechnology Co., Ltd., Wuhan, China) according to
the manufacturer's protocol.
Oxidative Stress Assay
Serum malondialdehyde (MDA, batch No. A003-1)
concentrations and superoxide dismutase (SOD,
batch No. A003-1) activity, which reflect the level of
oxidative stress, were measured using assay kits
(Elabscience Biotechnology Co., Ltd., Wuhan, China)
• 986 • Chin J Integr Med 2022 Nov;28(11):983-991

according to the manufacturer's protocol. GraphPad Prism version 7.0 (software, USA). The
measurement data are presented as the mean±
Fe2+ Concentration Analysis standard deviation ( x– ± s ). Differences among
Heart tissues were harvested and homogenized to groups were compared by one-way ANOVA or two-
measure the Fe2+ concentration using commercial kits way ANOVA followed by a Bonferroni post hoc test.
(batch No. A039-2, Elabscience Biotechnology Co., Ltd., P values less than 0.05 were defined as statistically
Wuhan, China) according to the manufacturer's protocol. significant.

Western Blot
RESULTS
Western blot was performed as described Effects of SMJ on Myocardial I/R Injury in Rats
previously. (2) Heart tissues were collected and As shown in Figures 1A–1C, the I/R group
homogenized with radioimmunoprecipitation assay showed a significant increase in myocardial infarct
lysis buffer, and total protein concentrations were size and CK-MB and cTnI release after ischemia
determined using a bicinchoninic acid kit (Beyotime compared with the sham group. SMJ-L group slightly
Bio, China). Equal amounts of protein were separated reduced myocardial I/R injury, as evidenced by the
by sodium dodecylsulfate/polyacrylamide gel decreases in myocardial infarct size and CK-MB
electrophoresis (5%–15% gel) and electrotransferred and cTnI release, but these decreases were not
onto polyvinylidene difluoride (PVDF) membranes. statistically significant (P >0.05). However, these
The PVDF membranes were incubated with primary indices were notably decreased in the SMJ-M and
antibodies against Nrf2 (1:600, 16396-1-AP, Thermo SMJ-H groups compared with the I/R group (P <0.05).
Fisher Scientific Inc. USA), GPX4 (1:1000, 14432-1-AP, There were no differences in myocardial infarct size
Thermo Fisher Scientific Inc. USA), and acyl-CoA or CK-MB and cTnI release between the SMJ-M and
synthetase long-chain family member 4 (ACSL4, SMJ-H groups (P >0.05).
1:1,000, PA5-30026, Thermo Fisher Scientific Inc.
USA) overnight at 4 ℃, followed by incubation with the Myocardial micromorphology was also observed
appropriate secondary antibody (1:10,000 dilution) for by HE staining (Figure 1D). The myocardial tissue in
45 min at room temperature. GAPDH (glyceraldehyde- sham group was dense and tidy, and the myocardial
3-phosphate dehydrogenase) was used as the loading fibres were intact without breakage. The myocardial
control. The immunoblot assays were replicated tissue in the I/R group showed severely disordered
3 times for each protein with myocardial tissue samples cellular arrangement, and the myocardial fibres were
(n =6). Signals were measured and quantified using a swollen and broken. The pathological alterations in the
fluorescence imaging scanner (Odyssey, USA). myocardium in the SMJ-L group were not markedly
different, and the degree of myocardial tissue lesions
Statistical Analysis in the SMJ-M and SMJ-H groups were significantly
All statistical analyses were performed with reduced compared with those in the I/R group.

A 50
B 1500
C 500

40 400
CK-MB release (U/L)

cTnl release (ng/L)

Infarct size (%)

△ △ △
△ 1000
30 △ △ 300

20 200
500
10 100

0 0 0
Sham I/R SMJ-L SMJ-M SMJ-H Sham I/R SMJ-L SMJ-M SMJ-H Sham I/R SMJ-L SMJ-M SMJ-H

D Sham I/R SMJ-L SMJ-M SMJ-H

Figure 1. Effects of Different Doses of SMJ on Myocardial I/R Injury in Rats ( ±s , n =6)
Notes: A: infarct area relative to the area at risk (IA/AAR×100%); B: serum CK-MB levels; C: serum cTnI levels; D: myocardial
micromorphology showed by HE staining under a light microscope (10×40); P <0.05 vs . sham group, △P <0.05 vs . I/R group
Chin J Integr Med 2022 Nov;28(11):983-991 • 987 •

500 200 8000 6000

I/R
SMJ-L

+dp/dt (mm Hg/s)

400

-dp/dt (mm Hg/s)

HR (Beats/min)

LVSP (mm Hg)

150 6000 △△
△ △ 4000 △△ SMJ-M
300 △△ SMJ-H
100 4000
200
2000
50 2000
100

0 0 0 0
e

ia

ia

ia

ia

n
in

in

in

in

o
m

m
si

si

si

si
sl

sl

sl

sl
he

he

he

he
fu

fu

fu

fu
Ba

Ba

Ba

Ba
er

er

er

er
sc

sc

sc

sc
ep

ep

ep

ep
ri

ri

ri

ri
te

te

te

te
rr

rr

rr

rr
Af

Af

Af

Af
te

te

te

te
Af

Af

Af

Af
Figure 2. Haemodynamic Parameters of Left Ventricular Function of
Rats in Each Group at Different Time Points ( ±s , n =6)
Notes: HR: heart rate; LVSP: left ventricular systolic pressure; ±dp/dt: maximal rates of left ventricular-developed pressure
increases and decreases; P <0.05 vs . baseline in the same group, △P <0.01 vs . I/R group at the same time point

As shown in Figure 2, compared with the 200 △ 2.0

△
△
baseline level, the haemodynamic parameters

MDA (nmol/mgprot)
150 1.5

SOD (U/mgprot)
△

significantly decreased after I/R injury in all groups 100 1.0

(P <0.05). Compared with I/R group, the HR, LVSP, 50 0.5

and ±dp/dt at the end of reperfusion in SMJ-L 0 0

Sham I/R Erastin Fer-1 Sham I/R Erastin Fer-1
group were slightly increased (P >0.05), while these
ACSL4 79 kD
parameters were significantly increased in SMJ-M and GAPDH 37 kD
SMJ-H groups (P <0.05). The data showed that SMJ-L 0.5
△
1.0 △

was not adequate to protect the myocardium from 0.4 0.8

Fe2+ (mg/kprot)

I/R injury, however, SMJ-M and SMJ-H groups had △

ACSL4/GAPDH
0.3 0.6
△
0.2 0.4
almost the same protective effects; thus, SMJ-M was
0.1 0.2
used in further experiments.
0 0
Sham I/R Erastin Fer-1 Sham I/R Erastin Fer-1

Ferroptosis Is Involved in Myocardial I/R Injury Figure 3. Ferroptosis Is Involved in Myocardial

I/R Injury in Rats ( ±s , n =6)
As shown in Figure 3, compared with the sham Notes: P<0.05 vs. sham group; △P<0.05 vs. I/R group
group, myocardial SOD activity in the I/R group was
significantly decreased accompanied by prominent solidified, density increased, and mitochondrial cristae
increases in MDA release, Fe 2+ concentration and decreased or disappeared. Pretreatment with Fer-1
ACSL4 protein expression (P <0.05). Compared with reduced damage to mitochondrial ultrastructure, while
the I/R group, the ferroptosis inhibitor Fer-1 but not erastin combined with I/R injury caused additional
the ferroptosis agonist erastin significantly increased damage to mitochondrial ultrastructure (Figure 4B).
SOD activity and decreased MDA release, Fe 2+ A Sham I/R Erastin Fer-1
concentration and ACSL4 expression in myocardial
I/R injury (P <0.05).
(200×)

As shown in Figure 4A, the myocardial fibers

were tidy and dense in the sham group. Compared B Sham I/R Erastin

with the sham group, the cellulars were edemaous and

(10,000×)

the myocardial fibers were ruptured and disordered in

the I/R group. In Erastin group, the myocardial fibers
showed more severely disordered, and the myocardial
fibres were swollen and broken; while the myocardial Fer-1 SMJ ML385+SMJ

tissue lesions in the Fer-1 group were significantly

(10,000×)

reduced. In addition, the ultrastructure of myocardial

mitochondria was observed by TEM. In the sham
group, the myocardial fibres were neatly arranged,
the mitochondrial membrane was complete, and the
Figure 4. Observation of Myocardial Morphology
cristae were clear. After I/R injury, mitochondrial size under Light Microscope (A) and Myocardial
significantly decreased, the mitochondrial membrane Ultrastructure under Electron Microscope (B)
• 988 • Chin J Integr Med 2022 Nov;28(11):983-991

50 2000 600

CK-MB release (U/L)

cTnl release (ng/L)

40

Infarct size (%)

1500
400
30
1000
20
200
500
10
0 0 0
I/R Erastin Fer-1 I/R Erastin Fer-1 I/R Erastin Fer-1
ACSL4 79 kD
GAPDH 37 kD
250 2.0 0.5 1.0
MDA (nmol/mg prot)

ACSL4/GAPDH
△ △
SOD (U/mg prot)

Fe2+ (mg/g prot)

200 1.5 0.4 0.8
△
150 0.3 0.6
1.0
100 0.2 0.4
50 0.5 0.1 0.2
0 0 0 0
I/R

tin

I/R

tin

I/R

tin

I/R

tin
SM

SM

SM

SM
as

as

as

as
Er

Er

Er

Er
J+

J+

J+

J+
SM

SM

SM

SM
Figure 5. Ferroptosis Is Involved in Myocardial I/R Injury, and SMJ Suppressed
Ferroptosis during Myocardial I/R Injury ( ±s , n =6)
Notes: P <0.05 vs . I/R group, △P <0.05 vs . SMJ group

SMJ, as well as Fer-1 Attenuated Myocardial I/R Nrf2 110 kD GPX4 32 kD

GAPDH 37 kD
Injury GAPDH 37 kD

Compared with the I/R group, the myocardial 1.5 1.0

△ 0.8 △
infarction size and CK-MB and cTnI release in the

GPX4/GAPDH
Nrf2/GAPDH

1.0
▲ 0.6 ▲
Fer-1 group notably decreased (P <0.05), whereas 0.4
0.5
those indice in Erastin group were further increased 0.2

(P <0.05, Figure 5). Moreover, 9 mL/kg SMJ significantly 0 0

am

I/R

85

am

I/R

85
attenuated myocardial I/R injury; While these
SM

SM
L3

L3
Sh

Sh
M

M
J+

J+
cardioprotective effects were completely abolished
SM

SM
by erastin treatment, as indicated by the increased Figure 6. Effects of SMJ with or without ML385 on
Activity of Nrf2/GPX4 Signalling ( ±s , n =6)
myocardial oxidative stress levels, Fe2+ concentrations Notes: P <0.05 vs . sham group, △
P <0.05 vs . the I/R
and ACSL4 expression (P <0.05, Figure 5). group, ▲P <0.05 vs . SMJ group

Effects of SMJ on Nrf2/GPX4 Signalling during of ginseng and Ophiopogon. Its active ingredients
Myocardial I/R Injury include ginsenosides Rbl, Rb3, and RgⅠ and
As shown in Figure 6, compared with the Ophiopogon polysaccharide, and SMJ has been
sham group, I/R injury significantly decreased the proven to have strong effects on the scavenging of
expression of Nrf2 and GPX4 (P <0.05). Pre-treatment oxygen free radicals and reducing lipid peroxidation.(20)
with SMJ markedly increased the protein expressions SMJ plays a key role in protecting against myocardial
of Nrf2 and GPX4 (P <0.05). I/R injury by upregulating the protein expression of
Bcl and downregulating the protein expression of
SMJ Attenuated Myocardial I/R Injury by Bax, which leads to a reduction in apoptosis.(13) In
Targeting Nrf2/GPX4-Mediated Ferroptosis the present study, the effects of SMJ on myocardial
As shown in Figure 7, compared with SMJ I/R injury and its optimal dose were examined. Our
group，the levels of cTnI，CK-MB and MDA notably data showed that SMJ could significantly attenuate
increased, while the level of SOD significantly myocardial I/R injury, and pretreatment with 9 mL/kg
decreased in SMJ+ML385 group (P <0.05). Moreover, SMJ was the optimal solution.
the Fe 2+ concentrations and ACSL4 expression in
SMJ+ML385 group noticeably increased than those Ferroptosis is a new type of PCD that was
in the SMJ group (P <0.05, Figure 7). discovered in 2012. It is iron-dependent and
characterized by lipid peroxidation damage caused
DISCUSSION by the excessive accumulation of reactive oxygen
SMJ is an injectable solution composed mainly species, which is completely different from other
Chin J Integr Med 2022 Nov;28(11):983-991 • 989 •

500 2000 300 △ 2.0

CK-MB release (ng/L)

cTnl release (ng/L)

MDA (U/mg prot)

SOD (U/mg prot)
400 1500 1.5
200
300
△ 1000 1.0
200 △
△
100
100 500 0.5

0 0 0 0
J

85

85

85

85

85

85

85

85
SM

SM

SM

SM
L3

L3

L3

L3

L3

L3

L3

L3
M

+M

+M

+M

+M
J+

J+

J+

J+
am

am

am

am
SM

SM

SM

SM
Sh

Sh

Sh

Sh
ACSL4 79 kD
GAPDH 37 kD
0.6 50 1.0
Fe2+ (mg/g prot)

ACSL4/GAPDH
Infarct size (%)
40 0.8
0.4
30 0.6

△ 20 0.4
0.2 △
10 0.2
0 0 0
J

85

85

85

85

85
SM

SM

SM
L3

L3

L3

L3

L3
M

+M

+M
J+

J+

J+
am

am
SM

SM

SM
Sh

Sh
Figure 7. Effects of SMJ with or without ML385 on Ferroptosis during Myocardial I/R Injury ( ±s , n =6)
Notes: P <0.05 vs . SMJ group, △P <0.05 vs . SMJ+ML385 group

types of PCD, such as apoptosis, pyroptosis, and sorafenib-induced ferroptosis. (31) GPX4 is the only
autophagy.(28) In recent years, ferroptosis has been gene mediated by the Nrf2 transcription pathway; it is
proven to be involved in a variety of pathophysiological a selenoprotein that can repair oxidative lipid damage
processes in mammals and is closely related to in cells in mammals and is the only enzyme that can
tumours, neurodegenerative diseases, diabetes, and reduce the lipid peroxides in biofilms. Studies have
cardiovascular diseases.(7) ACSL4 is involved in the shown that GPX4 knockout or inhibition significantly
synthesis of phosphatidyl ethanolamine, phosphatidyl increases intracellular ROS and accelerates lipid
inositol and other negatively charged membrane peroxidation in both the cell membrane and plasma
phospholipids and promotes ferroptosis by increasing membrane, ultimately leading to ferroptosis. (32)
the expression of lipotoxic 5-hydroxyeicosatetraenoic Accordingly, GPX4 is a key regulatory protein during
acid, which can be used as a specific marker of ferroptosis.(17) In the present study, the expression
ferroptosis.(29) In our present study, the concentration levels of Nrf2 and GPX4 were markedly decreased in
of myocardial Fe 2+, the release of MDA, and the the myocardium after I/R injury, which indicated that
expression of ACSL4, a specific ferroptosis protein, I/R injury impaired the activity and function of the
were noticeably increased after I/R insult in the Nrf2/GPX4 signalling pathway.
myocardium, and pretreatment with the ferroptosis
inhibitor Fer-1 but not the ferroptosis activator erastin The relationship between SMJ and Nrf2/GPX4
significantly reduced myocardial I/R injury. Our data signalling-mediated ferroptosis during myocardial I/R
were consistent with previous studies performed injury was further investigated. A recent study from
by Chen and Ma, et al(29,30) which strongly indicated Liu, et al (33) demonstrated that notoginsenoside
that ferroptosis was involved in the pathophysiology (NG)-R1, an active constituent of SMJ, could
of myocardial I/R injury and accounted for cardiac increase the activity of Nrf2. However, the effects of
dysfunction and myocardial damage. SMJ on Nrf2/GPX4 in myocardial I/R injury are still
unclear. Our data showed that SMJ pretreatment
Nrf2 plays an indispensable role as an significantly increased the expression of Nrf2 and
antioxidant, and the Nrf2-mediated antioxidant system GPX4 in I/R hearts, and the cardioprotective effects
resists exogenous and endogenous oxidative stress of SMJ were completely abolished by ML385, which
to maintain a dynamic balance.(14) Nrf2 has also been strongly indicated that SMJ activates the Nrf2/GPX4
reported to be an important negative regulator of pathway. In addition, we observed that SMJ disrupted
ferroptosis, and it was found that knocking out the ferroptosis in myocardial I/R injury, and this effect
Nrf2 gene in HCC cells could enhance erastin- or could be reversed by ML385. Thus, we showed that
• 990 •
Nrf2/GPX4 inactivation-induced ferroptosis was one
of the main causes of myocardial I/R injury and that
SMJ could activate the Nrf2/GPX4 pathway to inhibit
ferroptosis.
In the present study, compared with those in
the I/R group, post-treatment with SMJ maintained
myocardial mitochondrial membrane integrity,
prevented pyknosis, preserved mitochondrial
ultrastructure, reduced the levels of CK-MB, cTnI,
MDA, and ACSL4, and increased the expression of
Nrf2 and GPX4, indicating that SMJ could attenuate
myocardial ischemic reperfusion injury by inhibiting
iron-mediated cell death. ML385 is a Nrf2-specific
inhibitor, and in the present study, treatment with
ML385 significantly increased myocardial ischemia–
reperfusion injury, suggesting that it may inhibit the
Nrf2-GPX4 signalling pathway.
In conclusion, the present study provide
compelling evidence that Nrf2/GPX4 inactivation
contributes to ferroptosis-induced myocardial I/R injury
in rats. Pretreatment with 9 mL/kg SMJ significantly
attenuated myocardial I/R injury by restoring the
activity and function of Nrf2/GPX4 signalling and
disrupting ferroptosis.
Conflict of Interest
The authors declare that there are no conflicts of interest
regarding the publication of this paper.
Author Contributions
Xia ZY and Zhou B contributed to the experimental design,
supervision and were fund managers. Mei SL contributed to the
experimental design, implementation and writing of the paper.
Qiu Z, Jia YF and Zhou JJ participated in the experiments and
contributed to the data analysis.
REFERENCES
1. Jensen RV, Hjortbak MV, Bøtker HE. Ischemic heart
disease: an update. Semin Nucl Med 2020;50:195-207.
2. Heusch G. Myocardial ischaemia-reperfusion injury
and cardioprotection in perspective. Nat Rev Cardiol
2020;17:773-789.
3. Zhou B, Lei SQ, Xue R, Leng Y, Xia ZY, Xia ZY, et al.
DJ-1 overexpression restores ischaemic post-conditioning-
mediated cardioprotection in diabetic rats: role of
autophagy. Clin Sci (Lond) 2017;131:1161-1178.
4. Xue R, Lei SQ, Xia ZY, Wu Y, Meng QT, Zhan LY, et al.
Selective inhibition of PTEN preserves ischaemic post-
Chin J Integr Med 2022 Nov;28(11):983-991
conditioning cardioprotection in STZ-induced Type 1
diabetic rats: role of the PI3K/Akt and JAK2/STAT3
pathways. Clin Sci (Lond) 2016;130:377-392.
5. Qiu Z, Lei SQ, Zhao B, Wu Y, Su WT, Liu M, et al. NLRP3
inflammasome activation-mediated pyroptosis aggravates
myocardial ischemia/reperfusion Injury in diabetic rats. Oxid
Med Cell Longev 2017;9743280.
6. Dixon SJ, Lemberg KM, Lamprecht MR, Skouta R, Zaitsev
EM, Gleason CE, et al. Ferroptosis: an iron-dependent form
of nonapoptotic cell death. Cell 2012;149:1060-1072.
7. Tang M, Chen Z, Wu D, Chen L. Ferritinophagy/ferroptosis:
iron-related newcomers in human diseases. J Cell Physiol
2018;233:9179-9190.
8. Guiney SJ, Adlard PA, Bush AI, Finkelstein DI, Ayton S.
Ferroptosis and cell death mechanisms in Parkinson's
disease. Neurochem Int 2017;104:34-48.
9. Liu J, Guo ZN, Yan XL, Shuo H, Ren JX, Luo Y, et al.
Crosstalk between autophagy and ferroptosis and its
putative role in ischemic stroke. Front Cell Neurosci
2020;14:577403.
10. Zhou Z, Ye TJ, Decaro E, Buehler B, DeCaro E, Stahl Z,
et al. Intestinal SIRT1 deficiency protects mice from
ethanol-induced liver injury by mitigating ferroptosis. Am J
Pathol 2020;190:82-92.
11. Li DY, Liu B, Fan YM, Liu M, Han BH, Meng YX, et al.
Nuciferine protects against folic acid-induced acute
kidney injury by inhibiting ferroptosis. Br J Pharmacol
2021;178:1182-1199.
12. Lei G, Zhang Y, Koppula P, Liu X, Zhang J, Lin SH, et al.
The role of ferroptosis in ionizing radiation-induced cell
death and tumor suppression. Cell Res 2020;30:146-162.
13. Li WY, Li W, Leng Y, Xiong YH, Xia ZY. Ferroptosis is
involved in diabetes myocardial ischemia/reperfusion
injury through endoplasmic reticulum stress. DNA Cell Biol
2020;39:210-225.
14. He F, Ru XL, Wen T. NRF2, a transcription factor for stress
response and beyond. Int J Mol Sci 2020;21:13.
15. Shen YM, Liu XJ, Shi JH, Wu X. Involvement of Nrf2 in
myocardial ischemia and reperfusion injury. Int J Biol
Macromol 2019;125:496-502.
16. Yang WS, SriRamaratnam R, Welsch ME, Shimada K,
Skouta R, Viswanathan VS, et al. Regulation of ferroptotic
cancer cell death by GPX4. Cell 2014;1-2:317-331.
17. Forcina GC, Dixon SJ. GPX4 at the crossroads of lipid
homeostasis and ferroptosis. Proteomics 2019;18:1800311.
18. Ma CS, Lv QM, Zhang KR, Tang YB, Zhang YF, Shen Y,
et al. NRF2-GPX4/SOD2 axis imparts resistance to EGFR-
tyrosine kinase inhibitors in non-small-cell lung cancer cells.
Acta Pharmacol Sin 2021;42:613-623.
19. Gou ZX, Su XJ, Hu X, Zhou Y, Huang L, Fan Y, et al.
Chin J Integr Med 2022 Nov;28(11):983-991
Melatonin improves hypoxic-ischemic brain damage
through the Akt/Nrf2/Gpx4 signaling pathway. Brain Res
Bull 2020;163:40-48.
20. Shi L, Xie Y, Liao X, Chai Y, Luo Y. Shenmai Injection as an
adjuvant treatment for chronic cor pulmonale heart failure:
a systematic review and meta-analysis of randomized
controlled trials. BMC Complement Altern Med 2015;15:418.
21. Li L, Sha ZM, Wang YY, Yang DL, Li JH, Duan ZZ, et al.
Pre-treatment with a combination of Shenmai and Danshen
Injection protects cardiomyocytes against hypoxia/
reoxygenation- and H 2 O 2 -induced injury by inhibiting
mitochondrial permeability transition pore opening. Exp
Ther Med 2019;17:4643-4652.
22. Zhang S, You ZQ, Yang L, Li LL, Wu YP, Gu LQ, et al.
Protective effect of Shenmai injection on doxorubicin-
induced cardiotoxicity via regulation of inflammatory
mediators. BMC Complement Altern Med 2019;19:317.
23. Li L, Li JH, Wang QL, Zhao X, Yang DL, Liu L, et al.
Shenmai injection protects against doxorubicin-Induced
cardiotoxicity via maintaining mitochondrial homeostasis.
Front Pharmacol 2020;11:815.
24. Mei SL, Yu YL, Zeng HB, Lin GX. Effects of different
doses of Shenmai Injection on myocardial in rats. 2013
International Forum on Basic and Clinical Research
in Anesthesiology. Xi An, China. Medical and Health
Technology, 2013.
25. Zhang ZW, Wu Y, Yuan S, Zhang P, Zhang JY, Li HY,
et al. Glutathione peroxidase 4 participates in secondary
brain injury through mediating ferroptosis in a rat model of
intracerebral hemorrhage. Brain Res 2018;1701:112-125.
• 991 •
26. Hao SH, Yu J, He WM, Huang Q, Zhao Y, Liang BS, et al.
Cysteine dioxygenase 1 mediates erastin-induced
ferroptosis in human gastric cancer cells. Neoplasia
2017;19:1022-1032.
27. Sun J, Yu XH, Huangpu HQ, Yao FZ. Ginsenoside Rb3
protects cardiomyocytes against hypoxia/reoxygenation injury
via activating the antioxidation signaling pathway of PERK/
Nrf2/HMOX. Biomed Pharmacother 2019;109:254-261.
28. Yang WS, Stockwell BR. Ferroptosis: death by lipid
peroxidation. Trends Cell Biol 2016;26:165-176.
29. Chen X, Li J, Kang R, Klionsky DJ, Tang D. Ferroptosis:
machinery and regulation. Autophagy 2021;17:2054-2081.
30. Ma SX, Sun LY, Wu WH, Wu JL, Sun ZN, Ren JJ.
USP22 protects against myocardial ischemia-reperfusion
injury via the SIRT1-p53/ SLC7A11-dependent inhibition
of ferroptosis-induced cardiomyocyte death. Front Physiol
2020;11:551318.
31. Vanden BT, Linkermann A, Jouan-Lanhouet S, Walczak H,
Vandenabeele P. Regulated necrosis: the expanding
network of non-apoptotic cell death pathways. Nat Rev Mol
Cell Biol 2014;15:135-147.
32. Schriever SC, Zimprich A, Pfuhlmann K, Baumann P,
Giesert F, Klaus V, et al. Alterations in neuronal control of
body weight and anxiety behavior by glutathione peroxidase
4 deficiency. Neuroscience 2017;357:241-254.
33. Liu H, Yang JQ, Yang WQ, Hu SN, Wu YL, Zhao B, et al. Focus
on notoginsenoside R1 in metabolism and prevention against
human diseases. Drug Des Devel Ther 2020;14:551-565.
(Accepted June 24, 2022; First Online August 23, 2022)
Edited by ZHANG Wen