Biosci. Rep. (2016) / 36 / art:e00420 / doi 10.

1042/BSR20160082

Novel protective effects of pulsed

electromagnetic field ischemia/reperfusion
injury rats
Fenfen Ma*1 , Wenwen Li†‡1 , Xinghui Li†, Ba Hieu Tran§, Rinkiko Suguro§, Ruijuan Guan†, Cuilan Hou†,
Huijuan Wang||, Aijie Zhang†, Yichun Zhu† and YiZhun Zhu||¶2

*Department of Pharmacy, Shanghai Pudong Hospital, Fudan University, Shanghai 201399, China
†Shanghai Institute of Immunology & Department of Immunobiology and Microbiology, Shanghai Jiao Tong University School of Medicine,

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Shanghai 200025, China
‡Shanghai Key Laboratory of Bioactive Small Molecules and Research Center on Aging and Medicine, Department of Physiology and
Pathophysiology, Shanghai Medical College, Fudan University, Shanghai 200032, China
§Department of Pharmacology, School of Pharmacy, Fudan University, Shanghai 201203, China
||Longhua Hospital, Shanghai University of Tradition Chinese Medicine, Shanghai 201203,China
¶Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore 119228, Singapore

Synopsis
Extracorporeal pulsed electromagnetic field (PEMF) has shown the ability to regenerate tissue by promoting cell
proliferation. In the present study, we investigated for the first time whether PEMF treatment could improve the
myocardial ischaemia/reperfusion (I/R) injury and uncovered its underlying mechanisms.
In our study, we demonstrated for the first time that extracorporeal PEMF has a novel effect on myocardial I/R injury.
The number and function of circulating endothelial progenitor cells (EPCs) were increased in PEMF treating rats.
The in vivo results showed that per-treatment of PEMF could significantly improve the cardiac function in I/R injury
group. In addition, PEMF treatment also reduced the apoptosis of myocardial cells by up-regulating the expression of
anti-apoptosis protein B-cell lymphoma 2 (Bcl-2) and down-regulating the expression of pro-apoptosis protein (Bax).
In vitro, the results showed that PEMF treatment could significantly reduce the apoptosis and reactive oxygen species
(ROS) levels in primary neonatal rat cardiac ventricular myocytes (NRCMs) induced by hypoxia/reoxygenation (H/R). In
particular, PEMF increased the phosphorylation of protein kinase B (Akt) and endothelial nitric oxide synthase (eNOS),
which might be closely related to attenuated cell apoptosis by increasing the releasing of nitric oxide (NO). Therefore,
our data indicated that PEMF could be a potential candidate for I/R injury.
Key words: apoptosis, Bax, B-cell lymphoma 2 (Bcl-2), ischaemia/reperfusion (I/R) injury, pulsed electromagnetic
field (PEMF)

Cite this article as: Bioscience Reports (2016) 36, e00420, doi:10.1042/BSR20160082

INTRODUCTION process is closely related to postoperative complications [2,3]

caused by coronary artery vascular formation, coronary revascu-
larization and heart transplantation. After myocardium suffered
Hypertension, arrhythmia, myocardial infarction (MI) and severe ischaemia, restoration of the blood flow is a prerequisite
myocardial ischaemia/reperfusion (I/R) injury are all the most for myocardial salvage [2]. However, reperfusion may induce
common cardiac diseases, which are the major causes of mor- oxidative stress [4], inflammatory cell infiltration and calcium
tality in the world [1]. Among them, myocardial I/R injury is dysregulation [5]. All these players contribute to the heart dam-
the most important cause of cardiac damage. Its pathological age such as contraction and arrhythmias [6], generally named

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Abbreviations: Akt, protein kinase B; Bax, Bcl-2 associated X protein; Bcl-2, B-cell lymphoma 2; CCA, common carotid artery; CCK-8, Cell Counting Kit-8; CK, creatine kinase; CKMB,
creatine kinase isoenzyme-MB; DAPI, 4,6 -diamidino-2 -phenylindole; DHE, dihydroethidium; DMEM/F12, Dulbecco’s modified Eagle’s medium/F-12; dUTP, deoxyuridine triphosphate;
eNOS, endothelial nitric oxide synthase; EPCs, endothelial progenitor cells; flk-1, fetal liver kinase-1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HBDH, α-hydroxybutyrate
dehydrogenase; H/R, hypoxia/reoxygenation; HRP, horseradish peroxidase; I/R, ischaemia/reperfusion; LAD, left anterior descending; LDH, lactate dehydrogenase; MI, myocardial
infarction; MI/R, myocardial infarction/reperfusion; MI/RI, myocardial infarction/reperfusion injury; NRCMs, neonatal rat cardiac ventricular myocytes; PEMF, pulsed electromagnetic
field; ROS, reactive oxygen species; Sca-1, stem cell antigen-1; SD, Sprague Dawley; SHR, spontaneously hypertensive rats; TTC, 2,3,5-triphenyltetrazolium chloride; TUNEL, terminal
deoxynucleotidyl transferase-mediated dUTP nick-end labelling; VEGF, vascular endothelial growth factor.
1 These authors contributed equally to the article.
2 To whom correspondence should be addressed (email [email protected]).

c 2016 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution 1
Licence 4.0 (CC BY).
 F. Ma and others

myocardial I/R injury. Recently, more and more evolving ther- of physiology and pathology of Fudan University, Shanghai,
apies have been put into use for I/R injury. China).
Pulsed electromagnetic field (PEMF) is the most widely tested
and investigated technique in the various forms of electromag-
netic stimulations for wound healing [7], alleviating traumatic Myocardial I/R injury rat model and measurement
pain and neuronal regeneration [8,9].The rats were randomly di- of infarct size
vided into PEMF-treated (5 mT, 25 Hz, 1 h daily) and control All the rats were divided into three groups: (1) Sham: The silk
groups. They hypothesized the possible mechanism that PEMF was put under the left anterior descending (LAD) without liga-
would increase the myofibroblast population, contributing to tion; (2) I/R: Hearts were subjected to ischaemia for 45 min and
wound closure during diabetic wound healing. It is a non-invasive then reperfusion for 4 h; (3) I/R + PEMF: PEMF device was
and non-pharmacological intervention therapy. Recent studies in- provided by Biomobie Regenerative Medicine Technology. The
dicated that PEMF also stimulated angiogenesis in patients with I/R rats were pre-exposed to active PEMF for 2 cycles per day
diabetes [10], and could improve arrhythmia, hypertension and (8 min per cycle), whereas other two groups were housed with in-

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MI [1]. The MI rats were exposed to active PEMF for 4 cycles per active PEMF generator. I/R was performed by temporary ligation
day (8 min/cycle, 30 +− 3 Hz, 6 mT) after MI induction. In vitro, of the LAD coronary artery for 45 min through an incision in the
PEMF induced the degree of human umbilical venous endothelial fourth intercostal space under anaesthesia [11]. Then, the ligature
cells tubulization and increased soluble pro-angiogenic factor se- was removed after 45 min of ischaemia, and the myocardium was
cretion [VEGF and nitric oxide (NO)] [7]. However, the role reperfused for 4 h. Ischaemia and reperfusion were confirmed and
of PEMF in ischaemia and reperfusion diseases remains largely monitored by electrocardiogram (ECG) observation. The suture
unknown. Our study aimed to investigate the effects of PEMF was then tightened again, and rats were intravenously injected
preconditioning on myocardial I/R injury and to investigate the with 2% Evans Blue (Sigma–Aldrich). After explantation of the
involved mechanisms. hearts, the left ventricles were isolated, divided into 1 mm slices,
In our study, we verified the cardioprotective effects of and subsequently incubated in 2% 2,3,5-triphenyltetrazolium
PEMF in myocardial I/R rats and the anti-apoptotic effects chloride (TTC; Sigma–Aldrich) in 0.9% saline at 37 ◦ C for
of PEMF in neonatal rat cardiac ventricular myocytes (NR- 25 min, to distinguish infarcted tissue from viable myocardium.
CMs) subjected to hypoxia/reoxygenation (H/R). We hypothes- These slices were flushed with saline and then fixed in 10% par-
ized that PEMF treatment could alleviate myocardial I/R injury aformaldehyde in PBS (pH 7.4) for 2 h. Next, the slices were
through elevating the protein expression of B-cell lymphoma 2 placed on a glass slice and photographed by digital camera, the
(Bcl-2), phosphorylation of protein kinase B (Akt). Meanwhile, ImageJ software (NIH) was used in a blind fashion for analysis.
it could decrease Bax. We emphatically made an effort to in- Infarct size was expressed as a ratio of the infarct area and the area
vestigate the MI/R model and tried to uncover the underlying at risk [12].
mechanisms.

Pulsed electromagnetic field treatment

PEMF were generated by a commercially available healing
MATERIALS AND METHODS device (length × width × height: 7 cm × 5cm × 3cm) pur-
chased from Biomoble Regenerative Medicine Technology. The
Animals adapter input voltage parameter is approximately 100–240 V
Male, 12-week-old Sprague Dawley (SD) rats (250–300 g) and output parameter is 5 V. Fields were asymmetric and con-
were purchased from Shanghai SLAC Laboratory Animal. An- sisted of 4.5 ms pulses at 30 +
− 3 Hz, with an adjustable magnetic
imals were housed in an environmentally controlled breed- field strength range (X-axis 0.22 +− 0.05 mT, Y-axis 0.20 +
− 0.05
ing room and given free access to food and water supplies. mT, Z-axis 0.06 + − 0.02 mT).The I/R rats were housed in cus-
All animals were handled according to the “Guide for the tom designed cages and exposed to active PEMF for 2 cycles
Care and Use of Laboratory Animals” published by the US per time (8 min for 1 cycle), whereas the I/R rats were housed
National Institutes of Health (NIH). Experimental procedures in identical cages with inactive PEMF generator. For in vitro
were managed according to the Institutional Aminal Care study, culture dishes were directly exposed to PEMF for 1–2
and Use Committee (IACUC), School of Pharmacy, Fudan cycles as indicated (8 min for 1 cycle, 30 Hz, X-axis 0.22 mT,
University. Y-axis 0.20 mT, Z-axis 0.06 mT) [1]. The background magnetic
field in the room area of exposure animals/samples and controls
is 0 mT.
The measurement of blood pressure in SHR rats
At the end of 1 week treatment with PEMF, the rats were
anesthetized with chloral hydrate (350 mg/kg, i.p.), the right Detection of myocardium apoptosis
common carotid artery (CCA) was cannulated with poly- Terminal deoxynucleotidyl transferase-mediated dUTP nick-end
ethylene tubing for recording of the left ventricle pres- labelling (TUNEL) assay was applied to analyse cardiomyocyte
sures (MFlab 200, AMP 20130830, Image analysis system apoptosis. Heart samples were first fixed in 10% formalin and

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 Novel protective effects of pulsed electromagnetic field ischemia/reperfusion injury rats

then paraffin embedded at day 14. Then, the hearts were cut into Blots were rinsed three times (5 min each) with T-TBS and in-
5 μm sections. TUNEL staining was carried out as described cubated with horseradish peroxidase (HRP)-conjugated second-
previously [12]. When apoptosis occurred, cells would look ary antibody (1:10000, Proteintech) for 2 h at room temperature.
green. The blots were visualized by using enhanced chemiluminescence
(ECL) method (Thermo). GAPDH was applied to be the internal
control protein. Intensity of the tested protein bands was quanti-
Determination of myocardial enzymes in plasma fied by densitometry.
Blood samples were collected after haemodynamic meas-
urement and centrifuged at 3000 g for 15 min to get
the plasma. Creatine kinase (CK), lactate dehydrogenase Cell culture
(LDH), creatine kinase isoenzyme-MB (CKMB) and α- Primary neonatal rat cardiac ventricular myocytes (NRCMs)
hydroxybutyrate dehydrogenase (HBDH) were quantified by were collected as previously described [15]. Briefly, the ventricles
automatic biochemical analyzer (Cobas 6000, Roche). All of new born SD rats (1–3 days old) were minced and digested

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procedures were performed according to the manufacturer’s with 0.125% trypsin. Isolated cardiomyocytes were cultured in
protocols. Dulbecco’s modified Eagle’s medium/F-12 (DMEM/F12, Life
Technologies) supplemented with 10% (v/v) FBS (Life Tech-
nologies), 100 units/ml penicillin and 100 mg/ml streptomycin.
Myocardium cells morphology via TEM The following experiments used spontaneously beating cardi-
At the end of the experiment, sections from myocardial samples omyocytes 48–72 hours after plating. (37 ◦ C with 5% CO2 ).
of left ventricular were immediately fixed overnight in glutaral-
dehyde solution at 4 ◦ C and then incubated while protected from
light in 1% osmium tetroxide for 2 h. After washing with distilled
Cell treatment (hypoxia/reoxygenation)
NRCMs were prepared according to the methods recently de-
water for three times (5 min each), specimens were incubated in
scribed [15]. To establish the H/R model, the cells were cultured
2% uranyl acetate for 2 h at room temperature and then dehyd-
in DMEM/F-12 without glucose and serum. The cells were ex-
rated in graded ethanol concentrations. Finally, sections were
posed to hypoxia (99% N2 + 5% CO2 ) for 8 h, followed by
embedded in molds with fresh resin. The changes in morphology
reoxygenation for 16 h. The cells were pretreated with PEMF for
and ultrastructure of the myocardial tissues were observed and
30 min before the H/R procedure. The control group was cultured
photographed under a TEM [13].
in DMEM/F-12 with low glucose (1000 mg/l) and 2% serum un-
der normoxic air conditions for the corresponding times.
Scal-1 + /flk-1 + cells counting of endothelial
progenitor cells Cell viability assays
We applied antibodies to the stem cell antigen-1 (Sca-1) and fetal
The viability of NRCMs cultured in 96-well plates was measured
liver kinase-1 (flk-1) to sign endothelial progenitor cells (EPCs)
by using the Cell Counting Kit-8 (CCK-8) (Dojindo Molecular
as described before, and used the isotype specific conjugated
Technologies) according to the manufacturer’s instructions. The
anti-IgG as a negative control. The amount of Scal-1 + /flk-1 +
absorbance of CCK-8 was obtained with a microplate reader at
cells would be counted by flow cytometry technique [14].
450 nm.

Measurement of nitric oxide concentration and Measurement of intracellular reactive oxygen

Western blotting species levels
Plasma concentrations of NO were measured with Griess as- Reactive oxygen species (ROS) levels in NRVMs were determ-
say kit (Beyotime Institute of Biotechnology) according to the ined by dihydroethidium (DHE, Sigma–Aldrich) fluorescence
manufacturer’s protocol. The expressions of Bax, Bcl-2, p-Akt, using confocal microscopy (Zeiss, LSM 710). After differ-
Akt, p-endothelial nitric oxide synthase (eNOS), eNOS and ent treatments, cells were washed with D-PBS and incubated
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were as- with DHE (10 μmol/l) at 37 ◦ C for 30 min in the dark. Then, re-
sessed using Western blot as described recently [15]. Proteins sidual DHE was removed by PBS-washing. Fluorescent signals
were measured with Pierce BCA Protein Assay Kit (Thermo). were observed (excitation, 488 nm; emission, 610 nm) under a
Hippocampal protein lysates (50 mg/well) were separated us- laser confocal microscope (Zeiss).
ing (SDS/PAGE) under reducing conditions. Following elec-
trophoresis, the separated proteins were transferred to a PVDF
membrane (Millipore). Subsequently, non-specific proteins were Data analysis
blocked using blocking buffer (5% skim milk or 5% BSA in All the data were presented as means +
− S.E.M. Differences were
T-TBS containing 0.05% Tween 20), followed by overnight in- compared by one-way ANOVA analysis by using SPSS software
cubation with primary rabbit anti-rat antibodies specific for target version 19.0 (SPSS) and P value <0.05 was taken as statistically
proteins as mentioned before (Cell Signaling Technology) at 4 ◦ C. significant.

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Figure 1 The effect of PEMF on SHR rats in vivo. PEMF could lower the blood pressure in SHR rats. At day 7 treatment
with different intensity PEMF, blood pressure was recorded via CCA [1(A), 1(B), 1(C) and 1(D)]. Data were
represented as the mean + − S.E.M.
Differences were compared by one-way ANOVA analysis by using SPSS software version 19.0 (SPSS) and P value <0.05
was taken as statistically significant; (n=8–10 in each group).

RESULTS changes via CCA. We observed that PEMF treatment could sig-
nificantly lower the blood pressure in the Bioboosti WIN235 and
WI215-stimulating groups than that in non-treated ones (Fig-
PEMF could lower blood pressure under treatment ures 1A and 1B). But Bioboosti WIN221 and WC65 treating
of certain PEMF intensity in SHR rat model groups did not have any effects on the blood pressure in SHR
(double-blind) rats, compared with the non-treated ones (Figures 1C and 1D).
To determine whether PEMF has any effects on blood pressure Fields were asymmetric and consisted of 4.5 ms pulses at 30 +−3
of SHR rats, we treated SHR rats with different PEMF intensity Hz, with an adjustable magnetic field strength range (X-axis
1–4 cycles per day for 7 days and measured the blood pressure 0.22 +− 0.05 mT, Y-axis 0.20 +
− 0.05 mT, Z-axis 0.06 +− 0.02 mT).
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Figure 2 The effect of PEMF on the number of Scal-1 + /flk-1 + cells after treating EPSc for 7 and 14 days. PEMF treat-
ment notably increased the number of Scal-1 + /flk-1 + cells after treating EPSc for 7 and 14 days. Data were
represented as the mean + − S.E.M.
Differences were compared by one-way ANOVA analysis by using SPSS software version 19.0 (SPSS) and P value <0.05
was taken as statistically significant; (n=10 in each group).

The I/R rats were housed in custom designed cages and exposed oedema and less cell death. In Group Sham, the ruptured muscu-
to active PEMF for 2 cycles per time (8 min for 1 cycle), whereas lar fibres, mitochondrial or intracellular oedema and dead cells
the I/R rats were housed in identical cages with inactive PEMF were not observed (Figure 3B). To further confirm protective ef-
generator. fect of PEMF, we measured the MI size by applying TTC and
According to this result, we chose Bioboosti WIN235 as our Evans Blue staining in all three groups. The MI area in I/R +
needed PEMF to carry out the following experiments. PEMF group could be reduced, compared with the model rats in
I/R group (Figure 3C).

PEMF treatment could observably improve the

abundance of EPCs In vivo, PEMF dramatically reduced cell apoptosis
Amplifying EPCs abundance and function is an active focus of induced by I/R injury
research on EPCs-mediated neovascularization after I/R. Thus, As H/R of cardiomyocytes contributed to cell death, we also
the number of circulating EPCs was identified by Sca-1/flk- detected the effect on myocardial apoptosis by using TUNEL
1 dual positive cells as described. We determined that PEMF kit, as shown in Figure 4(A). We uncovered that PEMF pre-
treatment could remarkably increase the number of Scal-1 + /flk- treating could dramatically decrease apoptosis of myocardial
1 + cells in peripheral blood at postoperative days 7 and 14 cells in I/R + PEMF group, compared with I/R group. In
(Figure 2). addition, we also found that PEMF treatment could signific-
antly increase the expression of anti-apoptosis protein Bcl-
2, p-eNOS and p-Akt and down-regulated the expression
Preliminary assessment of PEMF showed great of pro-apoptosis protein Bax in the heart tissue, as shown
protective effect against myocardial in Figure 4(B).
infarction/reperfusion injury (MI/RI) rat model
To examine the effect of PEMF on myocardial I/R, male SD rats
were divided into three groups: Sham, I/R and I/R + PEMF (2 The effect of PEMF on cell viability in neonatal rat
cycles per day, 8 min per cycle) per day until 28 days. We ob- cardiac ventricular myocytes
served that PEMF stimulation could significantly decrease four To further investigate whether PEMF has the same effect
plasma myocardial enzymes (LDH, CK, CKMB and HBDH) in in vitro, we simulated the I/R injury model in vitro. We
I/R rats (Figure 3A). Additionally, we found that pre-stimulating applied NRCMs and hypoxia incubator to mimic myocar-
PEMF could improve the cardiac morphology via TEM, com- dial I/R injury via H/R as described in the section ‘Materi-
pared with I/R + PEMF group. TEM revealed the rupture of als and Methods’. We found that PEMF treatment (2 cycles)
muscular fibres, together with mitochondrial swelling, and intra- could remarkably improve cell viability, compared with the
cellular oedema in Group I/R. The shape of nucleus was irreg- H/R group (Figure 5). For in vitro study, culture dishes
ular, with evidence of mitochondrial overflow after cell death. were directly exposed to PEMF for 1–2 cycles as indicated
Compared with Group I/R + PEMF, less muscular fibres were (8 min for 1 cycle, 30 +
− 3 Hz, X-axis 0.22 +
− 0.05 mT, Y-axis
ruptured, with mild swelling of mitochondria, mild intercellular +
0.20 − 0.05 mT, Z-axis 0.06 +
− 0.02 mT).
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Figure 3 Protective effect of PEMF on I/R rats in vivo. Plasma myocardial enzymes (LDH, CK, HBDH and CKMB) content
was quantified by automatic biochemical analyzer (A) (n=18 in each group). Changes on cardiac cell morphology
via TEM (B) (n=6 in each group). TTC-Evans Blue staining for MI area (C). Statistical analysis of the effect of
PEMF in reducing infarct size in a rat model of I/R (D). Data were represented as the mean + − S.E.M.
Differences were compared by one-way ANOVA analysis by using SPSS software version 19.0 (SPSS) and P value <0.05
was taken as statistically significant.

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Figure 4 Apoptotic cardiomyocyte was identified by TUNEL analysis, apoptotic cardiomyocyte appears green whereas
TUNEL-negative appears blue (A), photomicrographs were taken at ×200 magnification. Apoptosis-related pro-
tein Bcl-2, Bax, p-Akt level of different treatments, p-eNOS level of different treatments, which were measured
by Western blot analysis (B–G). Plasma concentrations of NO were measured by spectrophotometer assay (H)
(n=8 in each group). Data were represented as the mean + − S.E.M.
Differences were compared by one-way ANOVA analysis by using SPSS software version 19.0 (SPSS); P < 0.05, compared
with the MI/RI ones (n=8 in each group).

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 F. Ma and others

ferent PEMF intensity (8 min for 1 cycle, 30 + − 3 Hz, X-axis

0.22 +
− 0.05 mT, Y-axis 0.20 +
− 0.05 mT, Z-axis 0.06 +
− 0.02 mT)
1–4 cycles per day for 7 days. PEMF can lower blood pressure
under treatment of certain PEMF intensity in SHR rat model
(double-blind). (2) PEMF has a profound effect on improv-
ing cardiac function in I/R rat model. (3) PEMF plays a vital
role in inhibiting cardiac apoptosis via Bcl-2 up-regulation and
Bax down-regulation. (4) In vitro, PEMF treatment also has a
good effect on reducing ROS levels by Akt/eNOS pathway to
release NO and improving cell apoptosis in NRCMs subjected to
hypoxia.
Many previous studies showed that extracorporeal PEMF-
treated(5 mT, 25 Hz, 1 h daily) could enhance osteanagen-

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Figure 5 NRCMs viability measured by CCK-8 assay at the end esis, skin rapture healing and neuronal regeneration, suggest-
of the treatment for 72 h. PEMF treatment enhanced the cell
viability of hypoxia NRCMs. Data were represented as the mean
ing its regenerative potency [8,16,17]. And some research-
+ S.E.M.
−
ers had found that PEMF therapy (8 min/cycle, 30 + − 3 Hz,
Differences were compared by one-way ANOVA analysis by using SPSS 6 mT) could improve the myocardial infarct by activating
software version 19.0 (SPSS) and P value <0.05 was taken as statist-
ically significant; P < 0.05, compared with the H/R ones (n=17 in each
VEGF–Enos [18] system and promoting EPCs mobilized to
group). the ischaemic myocardium [1,19]. Consistent with the previ-
ous work, our present study demonstrated that PEMF ther-
apy could significantly alleviate cardiac dysfunction in I/R rat
model.
Specific-density PEMF could decrease intracellular
Recent evidence suggest that circulating EPCs can be mobil-
ROS levels of primary cardiomyocytes subjected to
ized endogenously in response to tissue ischaemia or exogenously
hypoxia/reperfusion
by cytokine stimulation and the recruitment of EPCs contributes
As shown in Figure 6(A), NRCMs that were subjected to H/R
to the adult blood vessels formation [19,20,21]. We hypothesized
increased significantly the ROS level, whereas the ROS level
that PEMF could recruit more EPCs to the vessels. To confirm
had been decreased in PEMF group (2 cycles), in contrast with
our hypothesis, we applied antibodies to the Sca-1 and flk-1 to
the H/R group. Representative images of the ROS level were
sign EPC. The results indicated that PEMF could remarkably in-
displayed in Figure 6(B). At the same time, we identified the
crease the number of EPCs in the PEMF group, compared with
effect on NRCMs apoptosis after suffering H/R by using TUNEL
the I/R group.
kit. As shown in Figure 6(C), cell apoptosis in the H/R group
Previous evidence indicated that when heart suffered I/R,
was aggravated, whereas PEMF treatment could reduce the cell
cardiac apoptosis would be dramatically aggravated [22–24].
death. Representative images of TUNEL staining were shown in
Myocardial apoptosis plays a significant role in the pathogen-
Figure 6(D).
esis of myocardial I/R injury. We assumed that PEMF might
play its role in improving cardiac function through inhibiting cell
apoptosis. The Bcl-2 family is a group of important apoptosis-
Effect of PEMF on NO releasing via Akt/eNOS
regulating proteins that is expressed on the mitochondrial outer
pathway
membrane, endoplasmic reticulum membrane and nuclear mem-
Cultured NRCMs were treated with PEMF stimulation for 1 to 2
brane. Overexpression of Bcl-2 proteins blocks the pro-apoptosis
cycles and the supernatant and cell lysate were collected. When
signal transduction pathway, thereby preventing apoptosis caused
cells suffered H/R, intracellular levels of p-Akt, p-eNOS and
by the caspase cascade [25]. The role Bax plays in autophagy is a
Bcl-2 were decreased, whereas PEMF treatment could increase
debatable. Recently, new genetic and biochemical evidence sug-
the phosphorylation of Akt, p-eNOS and Bcl-2 (Figures 7A–
gest that Bcl-2/Bcl-xL may affect apoptosis through its inhibition
7C). The expression of Bax was increased when cells subjected
of Bax [26]. Overexpression of Bax protein promotes the apop-
to H/R whereas PEMF treatment reversed such increase (Fig-
tosis signal pathway. In the present study, we applied TUNEL
ure 7C). Western blot analysis was shown in Figure 7(D) for
staining to find that PEMF has a perfect effect on cardiac cell
p-Akt/Akt, Figure 7(E) for p-eNOS/eNOS, Figure 7(F) for Bcl-2
apoptosis by regulating apoptosis-related proteins Bcl-2 and Bax
and Figure 7(G) for Bax.
[25,26,27,28].
To verify our findings in the rat model, we mimicked I/R con-
dition in vitro by hypoxia exposure in NRCMs. Results showed
that not only in vivo, hypoxia could induce cell apoptosis in vitro.
DISCUSSION
And we also found that PEMF treatment could significantly alle-
viate cell apoptosis induced by hypoxia. At the basal level, ROS
Our present study provides the first evidence that PEMF has play an important role in mediating multiple cellular signalling
novel functions as follows: (1) We treated SHR rats with dif- cascades including cell growth and stress adaptation. Conversely,

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Figure 6 PEMF protected Neonatal rat cardiac ventricular myocytes (NRCMs) from hypoxia/reoxygenation (H/R)-induced
apoptosis via decreasing ROS levelat the end of the treatment for 72 h in vitro.
Effect of PEMF on ROS levels and apoptosis induced by hypoxia/reoxygenation in vitro. (A) Representative images of
ROS levels in NRCMs detected by confocal microscope. (B) Quantitative analysis for ROS levels in NRCMs were detected
by microplate reader. (C) TUNEL-positive nuclei quantification represented as number per high-power field (HPF). (D)
Representative photographs of cardiomyocyte apoptosis from NRCMs detected by confocal microscope-TUNEL (green),
apoptotic nuclei, DAPI (blue) and total nuclei. Data were represented as the mean +
− S.E.M. Differences were compared
by one-way ANOVA analysis by using SPSS software version 19.0 (SPSS) and P value <0.05 was taken as statistically
significant, (n=6 in each group).

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Figure 7 The related protein expression about the effect of PEMF on apoptosis induced by hypoxia/reoxygenationat
the end of the treatment for 72 h in vitro. PEMF increased the phosphorylation of Akt, endothelial nitric oxide
synthase (eNOS), and the expression of Bcl-2 anddown-regulated the expression of Bax.
The related protein expression about the effect of PEMF on apoptosis induced by H/R in vitro. PEMF could increase NO
releasing. (A, D) p-AKt level of different treatments. (B, E) p-eNOS level of different treatments. (C, F, G) Apoptosis-related
proteins Bcl-2, Bax, which were measured by Western blot analysis. Data were represented as the mean + − S.E.M.
Differences were compared by one-way ANOVA analysis by using SPSS software version 19.0 (SPSS) and P value <0.05
was taken as statistically significant, (n=8 in each group).

excess ROS can damage tissues by oxidizing important cellular In conclusion, this is the first study suggesting that PEMF
components such as proteins, lipids and DNA, as well as treatment could improve cardiac dysfunction through inhibiting
activating proteolytic enzymes such as matrix metallopro- cell apoptosis. Furthermore, in vitro, we first clarified PEMF still
teinases [29]. Previous studies showed that when cells were plays a profound effect on improving cell death and removing ex-
subjected to hypoxia, the intracellular ROS level would be cess ROS via regulating apoptosis-related proteins and Akt/eNOS
sharply increased, and the overproduction of ROS would res- pathway. All these findings highlight that PEMF would be applied
ult in cell damage [19,30,31]. In the present study, PEMF as a potentially powerful therapy for I/R injury cure.
treatment could prominently down-regulate ROS levels. We
also investigated how PEMF reduced the intracellular ROS AUTHOR CONTRIBUTION
level. Fenfen Ma designed and performed experiments on MI/RI rat
NO appears to mediate distinct pathways in response to oxid- model, histological stain and Western blot. Wenwen Li assisted
ative stress via AKt–eNOS pathway [32,33]. NO is identified as the in vivo experiments, validated the effect in vitro experiments,
gaseous transmitters. In vascular tissue, NO is synthesized from analysed data and wrote the manuscript. Xinghui Li interpreted data
L-arginine by nitric oxide synthase (NOS) and it is considered to and formatted manuscript. Rinkiko Suguro, Ruijuan Guan, Cuilan
be the endothelium-derived relaxing factor. Evidence show that Hou, Huijuan Wang and Aijie Zhang interpreted data and edited
the NO generation in endothelium cells was damaged in hyper- manuscript. Yichun Zhu and YiZhun Zhu proposed the idea and
tensive patients [34]. NO could also prevent platelet activation supervised the project.
and promote vascular smooth muscle cells proliferation [35]. NO
generation from eNOS is considered to be endothelium-derived
relaxing and ROS-related factor [36,37]. Some researchers found ACKNOWLEDGEMENTS
that bradykinin limited MI induced by I/R injury via Akt/eNOS We thank all of the members of the Laboratory of Pharmacology of
signalling pathway in mouse heart [38]. And bradykinin inhibited Chen Y., Ding Y.J. for their technical assistance.
oxidative stress-induced cardiomyocytes senescence by acting
through BK B2 receptor induced NO release [39]. Such evid- FUNDING
ence indicated that Akt phosphorylation could activate eNOS,
This work was supported by the key laboratory program of the
which lead to NO releasing, and resulted in ROS reducing. In the
Education Commission of Shanghai Municipality [grant number
present study, we found that PEMF decreased ROS via Akt/eNOS
ZDSYS14005].
pathway.
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 Novel protective effects of pulsed electromagnetic field ischemia/reperfusion injury rats

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Received 17 March 2016/11 October 2016; accepted 17 October 2016
Accepted Manuscript online 25 October 2016, doi 10.1042/BSR20160082

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12 c 2016 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution
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