This study explored whether the compound Rhein can inhibit stress in the endoplasmic reticulum and mitochondria caused by acute myocardial infarction through activating the PI3K/Akt/ERK signaling pathway. A rat model of myocardial infarction was used. Rats treated with Rhein showed improved cardiac function and reduced cardiomyocyte apoptosis compared to untreated rats. Rhein was found to inhibit expression of endoplasmic reticulum and mitochondrial proteins associated with stress and apoptosis, while elevating expression of proteins in the PI3K/Akt/ERK pathway. The results suggest Rhein can protect against myocardial damage from infarction by reducing endoplasmic reticulum and mitochondrial dysfunction through this signaling pathway.

1. 59
OBJECTIVE: To explore whether Rhein can inhibit
acute myocardial infarction–induced endoplasmic retic-
ulum and mitochondrial stress through activating the
phosphatidylinositol 3-kinase/serine/threonine kinase/
extracellular signal regulated kinase (PI3K/Akt/ERK)
pathway.
METHODS: A rat model of myocardial infarction was
established. The cardiac function injury was detected
by echocardiography. The apoptosis of cardiomyocytes
was detected by transferase-mediated dUTP-biotin nick
end labeling (TUNEL) fluorescence staining. The en-
doplasmic reticulum–related proteins C/EBP homology
protein (CHOP), glucose-regulated protein 78 (GRP78),
and cysteine-containing aspartate-specific proteases 12
(caspase-12), as well as mitochondrial cytochrome c,
Bcl-2 associated X protein (Bax), and B-cell lymphoma
2 (Bcl-2) were detected by immunofluorescence staining.
Western blot assay was employed to detect the expression
of CHOP, GRP-78, activating transcription factor 6
(ATF-6), Bcl-2, Bax, AKT, and ERK proteins.
RESULTS: Rhein could effectively improve cardiac
dysfunction caused by acute myocardial infarction.
Rhein can effectively protect cardiomyocytes and reduce
apoptosis. Rhein significantly inhibited the expressions
of endoplasmic reticulum CHOP, GRP-78, and other
proteins; reduced the expressions of mitochondria-
associated proteins such as cytochromec, Bax, and Bcl-2;
elevated the expressions of p-AKT and p-ERK proteins;
and inhibited PARP expression in the myocardial infarct
area.
CONCLUSION: Rhein can significantly reduce apop-
tosis induced by acute myocardial infarction injury
in rats, which requires the activation of Akt and ERK
signaling pathways and the inhibition of endoplas-
mic reticulum stress and mitochondrial dysfunction-
associated proteins. (Anal Quant Cytopathol Histpa­
thol 2021;43:59–66)
Keywords: acute myocardial infarction, apoptosis,
caspase 12, endoplasmic reticulum, endoplasmic
Analytical and Quantitative Cytopathology and Histopathology®
0884-6812/21/4302-0059/$18.00/0 © Science Printers and Publishers, Inc.
Analytical and Quantitative Cytopathology and Histopathology®
Mechanism of Rhein Inhibiting Acute
Myocardial Infarction–Induced Endoplasmic
Reticulum and Mitochondrial Stress by
Activating PI3K/Akt/ERK Pathway
Shuyu Liu, M.M., Woruo Ye, M.M., Tao Pan, M.D., Jing Liu, M.D., and
Jianbin Gong, M.D.
From the Department of Cardiology, First Clinical Medical College of Nanjing University of Chinese Medicine, Nanjing; the Department
of Cardiology, Jinling Clinical Medical College of Nanjing University of Chinese Medicine, Nanjing; and the Department of General
Medicine, Jiangsu Hospital of Chinese Medicine, Nanjing, China.
Shuyu Liu is Attending Physician, Departments of Cardiology, First Clinical Medical College of Nanjing University of Chinese Medicine
and Jinling Clinical Medical College of Nanjing University of Chinese Medicine.
Woruo Ye is Attending Physician, Department of General Medicine, Jiangsu Hospital of Chinese Medicine.
Tao Pan is Chief Physician, First Clinical Medical College of Nanjing University of Chinese Medicine.
Jing Liu is Deputy Chief Physician, Department of Cardiology, Jinling Clinical Medical College of Nanjing University of Chinese Medicine.
Jianbin Gong is Chief Physician, Department of Cardiology, Jinling Clinical Medical College of Nanjing University of Chinese Medicine.
Shuyu Liu and Woruo Ye contributed equally.
Address correspondence to: Jianbin Gong, M.D., Department of Cardiology, Jinling Clinical Medical College of Nanjing University of
Chinese Medicine, Nanjing 210002, China (gongjianbin802202@163.com).
Financial Disclosure: The authors have no connection to any companies or products mentioned in this article.

2. reticulum stress, mitochondrial proteins, myocardi­
al diseases, Rhein.
Endoplasmic reticulum stress is often accompanied
by an increase in myocardial oxidative free radi­
cals.1 The intracellular redox balance is destroyed
by excess reactive oxygen species, which triggers
early apoptosis of the cells.2 A large number of
studies suggest that excessive production of reac­
tive oxygen species is one of the important factors
that lead to endoplasmic reticulum stress,3 and this
excess reactive oxygen species endoplasmic retic­
ulum stress is called “reactive oxygen-dependent
endoplasmic reticulum excitation.” Endoplasmic
reticulum stress plays a very important role in the
growth, differentiation, and apoptosis of most cells.
Currently there is evidence that the activation of
the PI3K/Akt and MEK/ERK pathways is benefi­
cial for protecting cells from apoptosis induced by
endoplasmic reticulum stress.4 The activation of
ERK1/2 and PI3K/AKT signaling pathways can
protect cardiomyocyte survival and reduce early
apoptosis. Moreover, after the administration of
certain drugs, whether the ERK1/2 pathway or
the PI3K/AKT pathway will be activated, this ac­
tivation can protect all cardiomyocytes and avoid
early apoptosis.
Rhein has certain effects of anti-tumor, anti­
bacterial, anti-diuretic, anti-diarrhea, and anti-
inflammatory, but the role of Rhein in myocardial
infarction has not been reported yet. The present
research investigated whether Rhein can activate
the PI3K/Akt/ERK pathway to inhibit the expres­
sions of endoplasmic reticulum and mitochondrial
stress-related proteins induced by acute myocardial
infarction, reduce early apoptosis, and thus protect
against myocardial damage.
Materials and Methods
Establishment and Grouping of Myocardial Infarction
Models
A total of 100 8-week-old Sprague Dawley rats
weighing 200–220 g each were randomly divided
into 3 groups (20 per group), as follows: a sham
operation group (Sham), a myocardial infarction
control group (MI), and a Rhein administration
group. After 3% isoflurane-induced anesthesia, the
rats were fixed on the plate. The depilation and
disinfection of the anterior chest was completed,
the skin was cut open, and the chest wall muscle
was bluntly separated. The chest was opened,
and the extruded heart was exposed out of the
chest. The anterior descending branch was ligated
at about 1.5 mm at the junction of the left atrial
appendage and the pulmonary artery cone. The
sham group received the same surgical procedure,
and the ligature line passed under the anterior
descending without ligation. Intramyocardial
in­
jection of bone marrow stem cells was per­
formed at 3 points in the infarct border zone
at a dose of 20 μL per rat and a cell volume of
1×106. The rats in the control group received the
same dose of phosphate-buffered saline (PBS).
Rhein was administered by injection at a dose of
5 mg/kg per day, and the administration time
was from the first day of establishment of the
myocardial infarction model to the 28th day after
surgery.
Rat Echocardiography
Echocardiography was used to dynamically mon­
itor cardiac function and structure at the 1st, 7th,
14th, 21st, and 28th days after surgery. The rats
were anesthetized with 4% paraformaldehyde and
then placed on the platform at room tempera­
ture, breathing normally. Transthoracic echocar­
diography was performed using an ultrasound
system SONOS-7500 with a 12 MHz phased array
transducer. At the horizontal position of the pap­
illary muscle, the long axis and the short axis of
the parasternal were observed respectively, and
the rat cardiac M-mode echocardiogram was ob-
tained. The corresponding left ventricular end-
diastolic diameter (LVEDd) and end-systolic di-
ameter (LVESd) were measured and recorded. Left
ventricular ejection fraction (EF) and short axis
shortening rate (FS) were calculated. Data were
measured in at least 3 consecutive cardiac cycle
ultrasound images for quantitative analysis.
TUNEL Fluorescent Staining
The 28d heart tissue sections were placed on a
rack and baked in a 60°C incubator for 3 hours.
The rack was placed successively into xylene I for
10 minutes and xylene II for 10 minutes. The rack
was soaked successively in 100%, 90%, 80%, and
70% ethanol for 5 minutes respectively. An ap-
propriate amount of 20 g/mL DNase-free protein­
ase K was added to the section tissues and then
baked in a 37°C incubator for 30 minutes. The
sections were washed 4 times in a dye bath con-
taining PBS, 5 minutes each time. A total of 20 μL
of fluorescent-stained droplets was added to the
sample to cover the heart tissue, and the sections
60 Analytical and Quantitative Cytopathology and Histopathology®
Liu et al

3. were placed in a wet box in a 37°C incubator. The
tissues were incubated for 1 hour in darkness.
The sections were washed 4 times in a wash tank
containing PBS, 5 minutes each time. The anti-
fluorescence quencher carried by the kit was used
for sealing, and then the sections were photo­
graphed under a fluorescence microscope.
Annexin V-FITC/PI Staining
Adherent and suspended cells were collected from
each group and washed twice with precooled PBS.
The supernatant was discarded. The cells were
resuspended in 1× Binding Buffer. The cells were
resuspended with 500 μL 1´ Binding Buffer to
adjust the concentration to 5×106/mL. Annexin
V-FITC was added to the cell suspension. The
cells were incubated at room temperature in dark­
ness for 10 minutes. 10 μL PI was added to the
cell suspension. The cells were in darkness for 10
minutes. Within 2 hours the flow cytometer was
used for detection.
Western Blot Determination of the Expressions of
Various Proteins
The tissues of each group treated for 28 days were
collected and then washed twice with PBS. A host
of 400 μL of cell lysate was added to each flask,
and then 40 μL phenylmethylsulfonyl fluoride
(PMSF) was added. The cell culture flask was
gently agitated and placed on ice for 10 minutes
to lyse the cells sufficiently. The cells were re-
peatedly aspirated with a sterile syringe, and the
lysed product was added to the EP tube. The EP
tube was ice-bathed for 30 minutes and then cen­
trifuged at 12,000 g for 15 minutes. The superna­
tant was transferred to a new EP tube. The tube
was added with 20 μL of protein buffer for every
100 μL and boiled for 5 minutes, and then stored
under −80°C for further use.
The above samples were obtained. The proteins
were separated by 12% SDS-PAGE electrophore­
sis. The separated protein bands were transferred
to the PVDF membrane by wet method and then
blocked at room temperature for 1 hour. The pri­
mary antibody was added (concentration 1:1000).
The cells were incubated overnight at 4°C. The
primary antibody was eluted. The cells were in-
cubated with the secondary antibody (1:1000) for
1 hour. The secondary antibody was washed off.
Color development and fixation were performed
by chemiluminescence. The expressions of various
proteins were determined.
Immunofluorescence Staining
The baked 28d tissue sections in the oven were
taken out and soaked in xylene I for 15 minutes,
followed by soaking for 10 minutes in xylene II.
After removing the rack from xylene, the sections
were soaked successively in absolute ethanol, 95%,
and 75% ethanol for 5 minutes respectively. The
sections were rinsed with PBS twice, 5 minutes
each time. For high-pressure antigen retrieval, the
tis­
sue sections were immersed in a dyeing tank
containing sufficient PBS for 2 times, 5 minutes
each time, to wash off the surface sodium citrate
buffer. The sections were placed in 3% H2O2 and
allowed to stand at room temperature for 30 min­
utes. Tissue sections were immersed in a dyeing
tank containing sufficient PBS for 2 times, 5 min-
utes each time. The sections were placed in the
PBS-diluted 1% bovine serum albumin (BSA)
blocking solution and then placed in an oven at
37°C for 30 minutes. The excess BSA was wiped
away slightly without rinsing. The correspond­
ing protein primary antibody was diluted to the
desired concentration with 1% BSA. The CHOP
and GRP78 primary antibodies were diluted at
1:200, the caspase-12 primary antibody was dilut­
ed at 1:1000, and the PARP primary antibody was
diluted at 1:1000. In the Sham operation group
the primary antibody was changed to PBS as a
negative control and then all the sections were
placed in a wet box at 4°C overnight. The sections
were taken out and washed 3 times with PBS, 5
minutes each time. The sections were incubated
with the corresponding fluorescent secondary an-
tibody (diluted with 1% BSA). The concentration
of CHOP secondary antibody is 1:100, the concen­
tration of GRP78 secondary antibody is 1:100, the
concentration of caspase-12 secondary antibody is
1:500, and the PARP secondary antibody is diluted
at a proportion of 1:300. The sections were placed
neatly in a wet box and incubated for 2 hours at
37°C in an incubator. The tissue sections were
immersed in a dyeing tank containing sufficient
PBS for 3 times, 5 minutes each time. An anti-
fluorescence quencher was added to the periphery
of the heart tissue to seal the sections. The photo­
graph was taken under a fluorescence microscope.
Statistical Methods
All data are expressed as mean±standard devia-
tion (mean±SD). The t test was used for compar­
ison between 2 groups, and the comparison be-
tween multiple groups (>2) was performed using
Volume 43, Number 2/April 2021 61
Rhein Inhibiting Acute Myocardial Infarction

4. one-way ANOVA. There was a statistical signifi­
cance at p<0.05. The data were analyzed by Graph­
Pad Prism 5.0 (GraphPad Software, San Diego,
California, USA).
Results
The Effect of Rhein on Echocardiography
Echocardiographic results showed that the recov­
ery of cardiac function in the Rhein group was
significantly better than that in the MI group. At
28 days after the operation the cardiac function
(EF and FS) of the Rhein group was significant­
ly improved as compared with that in the MI
group, and the ESV and EDV were significantly
reduced as compared with those in the MI group
(Figure 1).
Rhein Inhibiting Myocardial Apoptosis Induced by
Myocardial Infarction
In normal myocardial tissues there are essential­
ly no TUNEL-positive cells, indicating almost no
apoptosis in normal tissues. After MI injury, ob-
viously TUNEL-positive cells were found in the
injured area, suggesting that a large amount of
apoptosis occurred after infarction. After treat­
ment with Rhein, it was found to have signifi-
cant protective effect on cardiomyocytes. The
number of apoptotic (TUNEL-positive) cells was
significantly reduced as compared to that in the
infarcted area (Figure 2).
Western Blot Determining the Expressions of Various
Proteins
The expressions of Caspase cascade-associated
proteins in tissues of myocardial infarction lesions
were determined by western blot. The results
showed that the expressions of cleaved PARP and
cleaved caspase-3, caspase-9, and caspase-12 were
significantly increased in injured tissues, and Rhein
can effectively inhibit the expressions of these
apoptosis-related proteins (Figure 3).
Mitochondrial function–related proteins in myo­
cardial tissue from infarcted injury were also ex-
amined, and Rhein significantly reduced the ex­
pressions of these 3 mitochondria-associated pro­
teins: cytochrome c (cyt c), Bax, and Bcl-2 (Figure 4).
In order to confirm the inhibitory effect of Rhein
on endoplasmic reticulum stress in infarcted tis­
sues, the expressions of 3 endoplasmic reticulum–
associated proteins—GRP78, CHOP, and ATF-6—
were detected. After treatment with Rhein, it was
found that the expressions of the above 3 endo-
plasmic reticulum–related proteins were signifi­
cantly inhibited (Figure 5).
To further investigate the specific upstream
mechanism of Rhein inhibiting endoplasmic retic­
ulum stress and mitochondrial function–related
proteins, it is believed that PI3K/Akt and ERK1/2
pathways may play an important role in this pro­
cess, so the 2 downstream signaling pathways
were measured. The expressions of p-AKT and
62 Analytical and Quantitative Cytopathology and Histopathology®
Liu et al
Figure 1
Effect of Rhein on rat
echocardiography. aa = p<
0.01 vs. MI, a = p<0.05 vs.
MI, bb = p<0.01 vs. Rhein,
b = p<0.05 vs. Rhein.

5. p-ERK in PI3K/Akt and ERK1/2 pathways were
clearly elevated after Rhein treatment (Figure 6).
Fluorescence Staining Results
To further confirm the inhibitory effect of Rhein
on endoplasmic reticulum stress in infarcted tis­
sues, immunofluorescence staining was performed
on infarcted lesions to detect the expressions of
3 endoplasmic reticulum–associated proteins:
GRP78, CHOP, and caspase-12. There were very
rare expressions of GRP78, CHOP, and caspase-
12 in normal myocardial tissues. However, the
expressions of these 3 endoplasmic reticulum–
associated proteins were significantly increased af-
ter the injury. After Rhein treatment, it was found
that the expressions of the above 3 endoplasmic
reticulum–associated proteins were significantly
inhibited (Figure 7).
Annexin V-FITC/PI Detection of Apoptosis Rate
The results showed that Rhein can cause apoptosis
of glioma cells (Figure 8).
Discussion
A large number of studies have shown that oxida­
tive stress–induced endoplasmic reticulum stress
plays an important role in cell dysfunction and
apoptosis in myocardial infarction.5 In the cir­
cumstance of hypoxia and glucose deprivation,
the endoplasmic reticulum stress marker protein
CHOP is elevated enough to trigger cardiomyo­
cyte death.6 The accumulation of unfolded pro-
teins in the endoplasmic reticulum will further
lead to endoplasmic reticulum stress. In moderate
cases, it is a kind of self-protection of cells. Some
stresses that can cause apoptosis can also activate
the endoplasmic reticulum signaling pathway.7
Volume 43, Number 2/April 2021 63
Rhein Inhibiting Acute Myocardial Infarction
Figure 2
Results of Rhein inhibiting
cardiomyocyte apoptosis
induced by myocardial
infarction.
Figure 3 Effect of Rhein on the expressions of Caspase cascade-
associated proteins.
Figure 4 Effect of Rhein on the expressions of mitochondria-
associated proteins.

6. GRP78 protein is a molecular chaperone of the
endoplasmic reticulum. GRP78 is elevated in en-
doplasmic reticulum stress. The increase of GRP78
is also a classic indicator of endoplasmic reticu-
lum stress. It is also reported that endoplasmic
reticulum stress can lead to early apoptosis, while
CHOP and caspase-12 are considered to be endo­
plasmic reticulum stress-mediated cells, which are
landmark protein pathways for apoptosis.8 In the
study it was found that endoplasmic reticulum–
associated proteins such as GRP78, CHOP, ATF-6,
and caspase-12 were significantly increased after
acute infarction, while exogenous treatment with
Rhein significantly inhibited endoplasmic reticu­
lum stress and effectively prevented apoptosis in
cells. These results indicated that the protective
effect of Rhein on myocardial infarction injury is
related to the inhibition of endoplasmic reticulum
stress-related proteins.
Mitochondrial dysfunction caused by oxidative
stress plays a critical role in the survival of car­
diomyocytes. In cardiomyocytes, at least 30% of
the energy required for cell contractile activity
is produced by mitochondria through oxidative
phosphorylation metabolic pathways. Mitochon­
dria are very sensitive to changes in the cellular
environment. When the environment changes
greatly, it may rapidly change from maintaining
cells to promoting apoptosis. Therefore, mitochon­
drial dysfunction and loss of cardiomyocytes and
myocardial weakness are not surprising. Studies
have suggested that myocardial infarction can lead
to mitochondrial dysfunction and cell death. The
initiation of apoptosis is due to the permeabiliza­
tion of the external mitochondrial membrane and
the release of death precursor proteins such as
cytochrome c, Bcl-2, and Bax, etc.9,10
The PI3K/Akt and ERK1/2 pathways are the
2 major downstream signaling pathways for bFGF
in myocardial protection. The PI3K/Akt path-
way plays an important role in the survival of
cardio­
myocytes under various lesions. The acti­
vation of Akt can phosphorylate the apoptotic
precursor protein Bax to inhibit the onset of
early apoptosis.11 Some scholars believe that the
activation of the PI3K/Akt signaling pathway
is critical for Rhein to maintain cell survival in
myocardial infarction cells. In addition, studies
have suggested that the cardioprotective effect of
Rhein requires the activation of the ERK signal-
ing pathway.12 In the study it was found and con­
firmed that there is a direct relationship between
the protective effect of Rhein on myocardial in­
farction injury and the activation of Akt and ERK
pathways.
In conclusion, Rhein can significantly reduce
apoptosis induced by infarcted injury in rats,
which requires the activation of Akt and ERK
signaling pathways, as well as the inhibition of
endoplasmic reticulum stress and expression of
64 Analytical and Quantitative Cytopathology and Histopathology®
Liu et al
Figure 5 Effect of Rhein on the expressions of endoplasmic
reticulum proteins.
Figure 6 Expressions of PI3K/Akt and ERK1/2 in the injured area
after Rhein activation of myocardial infarction.

7. mitochondrial dysfunction–associated proteins. In
the research it was confirmed that Rhein is a po-
tential drug for the treatment of myocardial infarc­
tion, thus providing a part of the theoretical basis
Volume 43, Number 2/April 2021 65
Rhein Inhibiting Acute Myocardial Infarction
Figure 7
The effect of Rhein on the
expressions of GRP78, CHOP,
and caspase-12 proteins.
Figure 8
The results of Annexin V-FITC/
PI detection of apoptosis rate.

8. for the development of targeted drugs on endo­
plasmic reticulum and mitochondrial stress.
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