2. long-term persistence can cause dilated cardio-
myopathy, heart failure, and even sudden death.
Cardiac hypertrophy, as an independent risk fac-
tor for cardiovascular diseases such as heart fail-
ure, significantly increases the incidence and mor-
tality of heart failure and is even more harmful to
heart disease patients with a smoking habit and
hypercholesterolemia. Therefore, a more compre-
hensive study of the occurrence and development
of cardiac hypertrophy will help to provide a new
target for the prevention and treatment of heart
failure.4 The further clarification of the specific
molecules and signaling pathways that mediate
cardiac hypertrophy, as well as the mechanism of
the development of cardiac hypertrophy, can pro-
vide a scientific basis in finding new targets for the
prevention and treatment of cardiac hypertrophy.
Rutin is used to prevent and treat cerebral hem
orrhage, hypertension, retinal hemorrhage, pur-
pura, and acute hemorrhagic nephritis. However,
there are few studies on the use of rutin for treat-
ing cardiac hypertrophy. The present study aimed
to investigate the effect of rutin on Angiotensin II
(Ang II)–induced cardiomyocyte hypertrophy and
the inherent mechanism, which is innovative in a
certain way.
Materials and Methods
The Culture of Primary Cardiomyocytes
Neonatal Sprague-Dawley suckling rats were ob-
tained. The chest was opened aseptically, and the
heart was quickly removed and placed in 4°C
D-Hank’s solution. One-third to one-half of the
whole heart was cut off, and the rest was washed
once with PBS. The heart was put in the glass
bottle containing 0.1% trypsin and placed on a
shaker at a speed of 40 times/min and then placed
in the refrigerator for overnight shaking at 4°C.
The liquid in the glass bottle was aspirated and
discarded, and the culture medium and trypsin
were added in equal amounts; the digestion was
terminated, and the supernatant was discarded. A
total of 7 mL of type II collagenase was added to
the glass bottle, which was shaken at a speed of
80 times/min for 10 minutes in a 37°C water
bath. The supernatant was collected, then neutral-
ized with an equal volume of culture solution.
All the digested cells were collected and trans-
ferred to a 15 mL centrifuge tube, filtered through
a 200-mesh filter, and placed in a centrifuge. The
supernatant was discarded to prepare a single cell
suspension, which was then inoculated in a 100 mm
petri dish. After being incubated for 50 minutes,
the unattached cell sus
pension was transferred to
another 100 mm cell culture dish to incubate for
another 45 minutes under the same conditions. The
cell suspension was aspirated, and the concentra-
tion was adjusted according to the experimental
requirements. A host of 100 µL BrdU was added
to every 10 mL cell suspension to inhibit fibroblast
proliferation and increase myocardial cell purity.
The cardiomyocytes in the supernatant were col-
lected and the cells were counted. The cells were
respectively inoculated into a 60 mm culture dish
at a rate of 1.5×106 cells/mL, into a 96-well plate
at a rate of 3×105 cells/mL and into a 6-well plate
at a rate of 1×106 cells/mL. The cells were placed
in an incubator for 48 hours culture, and then the
solution was changed.
Establishment and Grouping of Hypertrophic Models
of Primary Cardiomyocytes
The Control Group. Normal cardiomyocytes were
cultured for 24 hours, then 24 hours in low glucose,
and the solution was changed once.
The Model Group. Normal cardiomyocytes were
cultured for 24 hours, then 24 hours in low-glucose
culture solution. The solution was added with Ang
II (1 μmol/L) and changed once after 48 hours,
and the relevant indicators were measured after
72 hours.
The Low-Dose Administration Group. Normal pri-
mary cardiomyocytes were cultured for 24 hours
and then cultured in low-glucose culture medium
for 24 hours. A total of 5 μM rutin was added.
After 30 minutes, Ang II (1 μmol/L) was added,
and the drug was added again in 24 hours. In 48
hours the solution was changed once, and 72 hours
after medication the relevant indicators were mea-
sured.
The Medium-Dose Administration Group. Normal pri-
mary cardiomyocytes were cultured for 24 hours,
then cultured in low-glucose medium for 24 hours.
A total of 10 μM rutin was added. After 30 min-
utes, Ang II (1 μmol/L) was added. The drug was
added once in 24 hours, the solution was changed
once in 48 hours, and 72 hours after administration
the relevant indicators were measured.
The High-Dose Administration Group. Normal pri-
mary cardiomyocytes were cultured for 24 hours
200 Analytical and Quantitative Cytopathology and Histopathology®
Wang et al
3. and then cultured in low-glucose culture medium
for 24 hours. A total of 20 μM rutin was added.
After 30 minutes, Ang II (1 μmol/L) was added.
The drug was added once in 24 hours. The solu-
tion was changed once after 48 hours, and 72 hours
after medication the relevant indicators were mea-
sured.
CCK-8 Method to Observe the Survival Rate of
Myocardial Cells
The cardiomyocyte suspension was adjusted to
a cell density of 1×104/mL and inoculated in a
96-well plate for 48 hours culture. The cells were
grouped as above. The medium of each group was
aspirated, and the medium containing the corre-
sponding concentration of drug was added to the
drug groups. The medium in the normal control
group was changed with the same volume of
medium. The cells were incubated for 24 hours.
The viability of cardiomyocytes of each group was
detected by CCK-8 method. The absorbance of
each well was measured, the cell viability of the
normal control group was set at 100%, and the
percentages of cardiomyocyte viability of the drug
groups were calculated accordingly.
Western Blot Detecting the Expressions of Ca MKII,
HDAC, Ca N, β-MHC, and NFAT-3 Proteins
Cells from each group were collected and washed
twice with PBS. Each flask was added with 400 μL
of cell lysate and then with 40 μL of 10 mmol/L
PMSF. The flask was gently agitated and placed
on ice for 10 minutes to make the cells lyse evenly.
The cells were repeatedly aspirated with a sterile
syringe, and the lysed product was added to an
EP tube. The EP tube was ice-bathed for 30 min
utes and centrifuged at 12,000 g for 15 minutes.
The supernatant was transferred to a new EP tube
and the protein concentration was quantified by
the protein labeling BC method. Following this,
20 μL of protein sample buffer (6’) was added at
every 100 μL in each tube, and the tubes were
boiled for 5 minutes and then stored under −80°C.
The above samples were obtained, and the pro-
teins were separated by 12% SDS-PAGE electro
phoresis. The separated protein bands were trans-
ferred to the PVDF membrane by wet method and
then blocked at room temperature for 1 hour. The
cells were incubated overnight at 4°C with the pri-
mary antibody (Ca MKII, HDAC, Ca N, β-MHC,
and NFAT-3 antibody concentrations, all 1:1000)
and then washed with PBST 3 times. The second-
ary antibody (1:1000) was added for 1 hour incu-
bation. The cells were washed with PBST 3 times.
Color development and fixation were conducted
by chemiluminescence. The expressions of each of
the above proteins were determined.
Determination of Ca2+ Concentration and Ca2+
ATPase Activity in Cardiomyocytes
Cardiomyocytes inoculated in a 6-well plate from
each group were taken out and the medium was
discarded. The cells were washed 3 times with
PBS. Calcium ion probe Fluo-8/AM was added.
The cells were placed in an incubator with 5% CO2
for 45–60 minutes under 37°C and then washed 3
times with PBS. Dynamic scanning was performed
using a laser confocal microscope. A small amount
of PBS was added, and 20 cells were randomly
selected in each group. The intracellular calcium
ion concentration was measured according to the
fluorescence intensity of calcium ions by a software
analysis system.
The primary cardiomyocytes inoculated in a 60
mm culture dish were taken out from each group.
After 72 hours of administration, the cells were
digested by trypsin to make the cells detach. The
trypsin was neutralized with an equal amount of
medium. After centrifugation, the supernatant was
removed, and a small amount of PBS solution was
added. The cells were repeatedly freeze-thawed,
the supernatant was aspirated, and the content of
phosphorus was determined by the disrupted cell
enzymatic reaction; the Ca2+ ATPase activity was
calculated according to the standard curve.
Immunofluorescence Staining for Myocardial Cell
Identification and Surface Area Measurement
The cardiomyocytes inoculated in the 6-well plate
were taken out, and the medium was discarded.
The cells were washed 3 times with PBS and fixed
with 4% paraformaldehyde for 15 minutes. After
permeabilization with 0.25% Triton X-100 for 10
minutes, the cells were added with donkey serum
and blocked at room temperature for 30 minutes.
Following this, the anti-cardiac troponin T anti-
body was added to incubate for 1 hour at room
temperature. Fluorescent secondary antibody was
added, and the cells were incubated for 1 hour
at room temperature. The cells were added with
l g/mL of DAPI for 3 minutes under darkness. A
small amount of PBS was added to each group.
Cardiomyocyte images were acquired by a laser
confocal instrument of the LSM 710. In each inde-
Volume 41, Number 6/December 2019 201
Effect of Rutin on Cardiomyocyte Hypertrophy
4. pendent experiment, 3 wells of each group were
obtained, and the experiment was repeated 3 times.
Five images from different perspectives were ran-
domly taken from each cell sample, and 10 cells
were obtained from each image; the cell surface
area in the 3 experiments was measured by Image-
Pro Plus 6.0 software.
Determination of Nitric Oxide (NO) Concentration
and NOS Activity in the Cell Supernatant
The cardiomyocytes of each group inoculated in
a 25 mm culture flask were removed, and after 72
hours of administration the cell supernatant was
collected. Both NO and NOS were determined by
colorimetric method. The reaction of NO2- with
Griess reagent was measured at 540 nm to gen
erate the absorbance value of cherry red. The NO
level per well can be calculated according to the
standard curve. At a wavelength of 530 nm, the
measured absorbance represents NOS activity,
which can be calculated based on the amount of
absorbance.
Statistical Methods
All data are expressed as mean±standard devia-
tion (mean±SD). The t test was used for compari-
son between the two groups, and the comparison
between multiple groups of data (>2) was per-
formed using one-way ANOVA. There was a statis-
tical significance at p<0.05. The data were analyzed
using GraphPad Prism 5.0 (GraphPad Software,
San Diego, California, USA).
Results
Effect of Rutin on Myocardial Cell Viability After
Ang II Treatment
The CCK-8 method was used to observe the
effect of different concentrations of rutin on the
activity of Ang II–induced cardiomyocytes. The
results showed that the cell viability in the Model
group decreased significantly. As compared with
the Model group, the cell viability in the drug-
pretreated groups increased, and in a concentration-
dependent manner (Figure 1).
Effect of Rutin on the Expressions of Ca MKII,
HDAC, Ca N, c-Jun, ANP, BNP, β-MHC, and
NFAT-3 Proteins
The results of western blot showed that in the
Model group the expressions of Ca MKII, HDAC,
Ca N, NFAT-3, c-Jun, ANP, and BNP proteins in
the cardiomyocytes increased. After drug admin-
istration, their expressions were significantly re-
duced, and in a concentration-dependent manner.
The results indicated that rutin can inhibit the
expressions of Ca MKII, HDAC, Ca N, NFAT-3,
c-Jun, ANP, and BNP proteins that were induced
by Ang II (Figure 2).
Effect of Rutin on the Surface Area of Hypertrophic
Cardiomyocytes Induced by Ang II
As compared with the Control group, the surface
area of myocardial cells in the Model group was
significantly increased. The drug could reduce the
surface area of myocardial cells and the myocar-
dial cell hypertrophy caused by Ang II. The fluo
rescence microscopy results of myocardial cell size
in each group are shown in Figure 3.
Effect of Rutin on Ca2+ Concentration and Ca2+
ATPase Activity in Myocardial Cells
As compared with the Control group, the Model
group exhibited significantly increased Ca2+ con-
centration and Ca2+ ATPase activity in the cardio-
myocytes. As compared with the Ang II groups,
after different doses of the drug and Ang II acted
together, the cardiomyocyte Ca2+ concentration
was significantly decreased, while the Ca2+ ATPase
activity was significantly increased, and both in
a concentration-dependent manner. The results
showed that rutin inhibited the increase of Ca2+
concentration and increased Ca2+ ATPase activ
202 Analytical and Quantitative Cytopathology and Histopathology®
Wang et al
Figure 1 Effect of rutin on the viability of cardiomyocytes
after Ang II treatment. *p<0.05 vs. model. **p<0.01 vs. model.
#p<0.01 vs. 5 μM. ##p<0.01 vs. 5 μM. &p<0.05 vs. 10 μM.
5. Volume 41, Number 6/December 2019 203
Effect of Rutin on Cardiomyocyte Hypertrophy
Figure 2 Effect of rutin on each protein. A=control group, B=model group, C=low-dose group, D=medium-dose group, E=high-dose
group. *p<0.05 vs. model group. **p<0.01 vs. model group. #p<0.05 vs. medium-dose group. ##p<0.01 vs. medium-dose group. &p<0.01
vs. low-dose group. &&p<0.01 vs. low-dose group. !p<0.05 vs. high-dose group.
Figure 3
Effect of different
concentrations of rutin on
Ang II–induced hypertrophic
cardiomyocytes. *p<0.05 vs.
model group. **p<0.01 vs.
model group. #p<0.05 vs.
medium-dose group. &p<0.01
vs. low-dose group. &&p<0.01
vs. low-dose group. !p<0.05
vs. high-dose group.
6. ity in cardiomyocytes induced by Ang II (Figures
4–5).
Effect of Rutin on NO Concentration and NOS
Activity in Ang II–Induced Hypertrophic
Cardiomyocytes
As compared with the Control group, the NO
concentration and NOS activity in cardiomyo-
cytes in the Model group were significantly de-
creased. After different doses of drug administra-
tion of the Model group, the NO concentration and
NOS activity in cardiomyocytes increased signifi-
cantly, and in a concentration-dependent manner
(Figures 6–7).
Discussion
Cardiac hypertrophy is a major risk factor for
cardiovascular disease in humans. It is mainly
characterized by increased myocardial cell protein,
increased cell volume, changes in interstitial com-
position, and decreased cardiac compliance and
circulatory function. The formation involves the
participation of multiple factors.5,6 Due to the oc-
currence of cardiac hypertrophy, the sarcoplasmic
reticulum calcium pump activity is reduced, caus-
ing calcium ion disorder. In the diastolic activity
of the heart, the cardiac sarcoplasmic reticulum
Ca2+ ATP plays an important role by ingesting
and releasing Ca2+. At the same time, the increase
of intracellular Ca2+ concentration is the central
link of the development of cardiac hypertrophy
caused by external stimuli and/or intrinsic func-
tional defects.7,8
On the one hand, the decrease of Ca2+ ATP
activity can cause calcium disorder, and calcium
overload is one of the main pathophysiological
mechanisms leading to myocardial systolic dys-
function, which causes cardiac hypertrophy. On
204 Analytical and Quantitative Cytopathology and Histopathology®
Wang et al
Figure 4 Effect of rutin on the concentration of Ca2+ ATP in
myocardial cells. *p<0.05 vs. model. **p<0.01 vs. model.
#p<0.01 vs. 5 μM. ##p<0.01 vs. 5 μM.
Figure 5
Effect of rutin on the
concentration of [Ca2+] in
hypertrophic cardiomyocytes
induced by Ang II. *p<0.05 vs.
model. **p<0.01 vs. model.
#p<0.01 vs. 5 μM. ##p<0.01
vs. 5 μM. &p<0.05 vs. 10 μM.
7. the other hand, the change of intracellular Ca2+
concentration caused by various circulating stim-
uli through different mechanisms can initiate the
corresponding signal transduction pathway, lead-
ing to cardiac hypertrophy, wherein the main cur-
rent of the action potential plateau is the calcium
current of the L-type calcium channel. The latter
function depends on channel protein phosphor-
ylation, while intracellular Ca2+ homeostasis can
decrease the phosphorylation level of L-type cal-
cium channels by Ca N, reduce Ca2+ influx, and
provide feedback regulation.9,10
Studies have shown that NO-cGMP-PKG in-
hibits L-type calcium channel Ca2+ influx, inhibits
Ca N activation, attenuates NFAT nuclear translo
cation and transcriptional activity, and is resistant
to cardiac hypertrophy. Ca2+/Ca M–dependent
protein kinase may play a role in cardiac hyper-
trophy by activating the cAMP-response element
binding protein (CREBP) transcription factor fam-
ily.11 The present study found that, as compared
with the Ang II–induced cardiac hypertrophy
model group, the drug can significantly reduce the
expressions of CaN, NFAT-3, CaMK, and HDAC
proteins in cardiomyocytes, suggesting that the in-
hibitory effect of drugs on Ang II–induced cardiac
hypertrophy may be related to the CaN-NFAT and
CaMKII-HDAC signal transduction pathway me-
diated by Ca2+.
As a vascular endothelial relaxing factor, NO is
a molecular messenger substance with multiple
functions, which binds to receptors on the cell
membrane, reduces Ca2+ efflux in smooth muscle
cells, relaxes blood vessels, and relieves cardiac
hypertrophy through corresponding signal trans-
duction pathways.12,13 The results of the experi-
ment showed that, as compared with the Model
group, the NO level in the drug group was higher.
The Ang II–induced cardiomyocyte hypertrophy
is inhibited by the drug, which can significantly
increase NO concentration in the supernatant of
cardiomyocytes and increase the activity of NOS.
The increase of coronary diastolic blood flow can
be realized through increasing the concentration
of NO in the heart, thus playing an important role
in cardiac hypertrophy. This suggests that the
drug’s protective effect on cardiomyocyte hyper-
trophy is possibly related to NO concentration in
the heart.
In conclusion, rutin can improve the survival
rate of cardiomyocyte hypertrophy induced by
Ang II, reduce the surface area of cardiomyocytes,
and decrease the proto-oncogene c-Jun and cardiac
hypertrophic factors ANP, BNP, and β-MHC. The
drug reduces the expressions of CaMKII, HDAC,
Ca N, and NFAT-3, decreases the concentration
of intracellular Ca2+, increases the activity of Ca2+
ATPase, and increases NO concentration and
Volume 41, Number 6/December 2019 205
Effect of Rutin on Cardiomyocyte Hypertrophy
Figure 6 Effect of rutin on NO concentration in Ang II–induced
hypertrophic cardiomyocytes. *p<0.05 vs. model. **p<0.01 vs.
model. #p<0.01 vs. 5 μM. ##p<0.01 vs. 5 μM. &p<0.05 vs. 10
μM. &&p<0.01 vs. 10 μM. !p<0.01 vs. 20 μM.
Figure 7 Effect of rutin on NOS activity in Ang II–induced
hypertrophic cardiomyocytes. *p<0.05 vs. model. **p<0.01 vs.
model. #p<0.01 vs. 5 μM. ##p<0.01 vs. 5 μM. &p<0.05 vs. 10
μM. !p<0.01 vs. 20 μM.
8. NOS activity in cells. The possible mechanism of
rutin is through blocking calcium-mediated CaN-
NFAT-3 and CaMKII-HDAC signaling transduc-
tion pathways.
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