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CARDIOVASCULAR JOURNAL OF AFRICA • Volume 31, No 1, January/February 2020
26
AFRICA
to regulate Nrf2 transcriptional activity and alter the cellular
distribution of Nrf2, while exhaustive exercise alters the levels of
p38MAPK and ERK. Therefore we hypothesised that exhaustive
exercise may impact on the heart by modulating Nrf2 expression.
Salidroside (Sal) is an effective extract obtained from
Rhodiola
rosea
that induces Nrf2 expression.
15,16
The intervention of Sal
improved little in normal rats, but Sal increased coronary flow,
improved cardiac function, reduced myocardial ischaemia–
reperfusion injury, and improved the myocardial ultrastructure
and energy metabolism in exhausted rats.
2,3,17
Sal altered
myocardial levels of proteins in the MAPK pathway.
18
MAPK
activation improved cardiac arrhythmia and other cardiac
diseases.
19,20
Therefore we hypothesised that the protective effect
of Sal on the exhausted heart was related to Nrf2 expression. In
these experiments, we aimed to explore the effects of exhaustive
exercise on myocardial levels of Nrf2 and Keap1.
Methods
Forty-eight male Sprague-Dawley rats (385
±
34 g) were provided
by the Academy of Military Medical Sciences (Beijing). National
standard rodent dry feed was provided
ad libitum
, the indoor
temperature was maintained at 18 to 22°C, and the relative
humidity was maintained at 40 to 55%.
All experiments were conducted in compliance with the Guide
for the Care and Use of Laboratory Animals and reviewed and
approved by the Ethics Committee for the Use of Experimental
Animals at No. 252 Hospital of the Chinese People’s Liberation
Army.
The main reagents used in this study are listed below. The
98% rhodionine powder was purchased from Nanjing Zelang
Pharmaceutical Technology Co, Ltd. Solutions containing
specific concentrations of Sal were generated by dissolving
the Sal powder in sterile normal saline. The cardiac troponin I
(cTnI), brain natriuretic peptide (BNP), CAT and GSH enzyme-
linked immunoassay kits were obtained from BD Biosciences
(New York, USA). The ROS enzyme-linked immunoassay kit
was purchased from R&D Systems (USA) and the anti-Nrf2
and anti-Keap1 antibodies were purchased from Abcam (UK).
The TRIzol total RNA extraction reagent was purchased from
Tiangen Biotech Co, Ltd. The PrimeScript™RT reagent kit with
genome DNA Eraser, SYBR
®
Premix Ex Taq™ II, DL2 and
DNA marker were purchased from TaKaRa Co, Ltd.
The following main instruments were used in this study: a
PowerLab signal acquisition and analysis system, MultiscanGO
enzyme standard instrument (Thermo, USA), Sigma 3k15
high-speed refrigerated centrifuge (Sigma, Germany), pressure–
volume catheter (SPR-838, Millar Company, USA), fluorescence
quantitative PCR platform (ABI 7500, Applied Biosystems),
vertical electrophoresis system (BIO-TEK, USA), transfer
electrophoresis system (BIO-TEK, USA), gel imaging system
(BioSpectrum), image analysis system (Image-Pro Plus 4.1),
PowerLab data acquisition and analysis system (AD Instruments,
Australia), bioelectric amplifier (AD Instruments, Australia) and
a needle electrode (AD Instruments, Australia).
Sprague-Dawley rats were randomly divided into four groups
(
n
=
12 rats per group): the control, an acute exhaustive
swimming group (ES), a low-dose Sal plus acute exhaustive
swimming group (SLE), and a high-dose Sal plus acute exhaustive
swimming group (SHE). Six of the 12 animals in each group
were used for the pressure–volume catheter detection of cardiac
function, which was an invasive experiment. These animals were
euthanised after the experiment.
Serum, electrocardiogram and myocardial specimens were
collected from the remaining animals (
n
=
6 rats per group).
Each group was administered the Sal solution (15 or 30 mg/kg/d)
or the same amount of normal saline for 14 days. The adaptive
swimming exercise was performed three times (20 min/time)
during the irrigation period. The control group did not exercise.
The rats in the ES, SLE and SHE groups were submitted
to one exhaustive swimming training session after the 14-day
treatment. Because eating would increase the time an animal
would be required to swim to reach exhaustion, rats were
fasted for 12 hours before training. The water temperature was
maintained at 32°C, and the temperature fluctuated by no more
than 1°C. Each rat in the exhaustion groups carried a tin wire
(3% body weight) on the tail. The exhaustive swimming exercise
was performed until exhaustion was achieved.
The experimental animal model of exhaustive exercise-induced
damage was established according to the standards described by
Thomas: animals were unable to return to the surface of the
water for 10 seconds and when placed upside down, they were
unable to complete a righting reflex.
21
Their fur was dried with a
heater immediately after exhaustion was reached.
Rats were subjected to abdominal anaesthesia with
pentobarbital sodium (40 mg/kg), the chest was opened, and
blood was collected from the inferior thoracic vena cava. The
blood was centrifuged at 3 000 rpm for 20 minutes and the
supernatant was collected and stored in a –80°C freezer until
detection of the serum indicators.
The hearts were quickly removed and washed with cold saline.
Tissues were stored individually at –80°C until q-PCR and
Western blot analysis was done.
Determination of cardiac function parameters with
a pressure–volume catheter
22
Rats were anaesthetised with pentobarbital sodium (40 mg/kg,
intraperitoneal), and the closed-chest approach was chosen for
catheter insertion. The animal was fixed in the supine position
on the operating table. The skin of the neck was disinfected
prior to a midline neck incision, and the trachea was separated
and intubated. The right carotid artery was separated from the
common carotid artery. Two 4-0 silk threads were sewn through
the common carotid artery, and one of the silk threads was used
to ligate the proximal end of the carotid artery. A cut was made
at the end of the heart to complete the knot.
The pressure–volume catheter was inserted through the
incision into the left chamber along the inverse blood flow of the
carotid artery and calibrated with MPVS control software. The
left ventricular pressure–volume waveform of the anaesthetised
rats was recorded with Chart7 software in real-time. Vessels and
catheters were fixed with another silk thread. Baseline data were
recorded for 15 minutes.
The abdominal skin was disinfected, a median incision was
made, the inferior vena cava was occluded, and changes in the
waveform were recorded. A 20-
μ
l solution of 30% NaCl was
rapidly injected into the anterior jugular vein and pressure–
volume waveform changes were recorded. The first four holes
of a calibration cuvette with known diameters (provided by