Extract
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Measurement of TNF-a Neonatal cardiomyocytes plated
at a density of 5×105 cells/mL were incubated with LPS
(O55:B5, Sigma) at concentrations of 1, 5, 10, and 20 µg/mL for 6 h.
In another experiment, the neonatal rat cardiomyocytes were
treated with 10 µg/mL LPS alone or in combination with 1, 2,
or 4 µmol/L neutral sulfate berberine (Sigma) for 6 h.
TNF-a concentrations in cell-conditioned media were measured by
using a Quantikine enzyme-linked immunosorbent assay kit
(R&D Systems, Minneapolis, USA) for rat TNF-a,
according to the manufacturerĄŻs instructions.
Fura-2 acetoxymethyl ester loading and measurement
of [Ca2+]i Neonatal rat cardiomyocytes
(5×105 cells/mL) were loaded with
fura-2/acetoxymethyl (AM) ester (Sigma) at a final concentration of 5 µmol/L for 30 min in the dark, then
the cells were washed with HanksĄŻ solution 3 times to
remove the extracellular fura-2/AM. Based on methods
described previously[12], Fura-2 fluorescence was monitored
with a Shimadzu RF-5000 fluorescence spectrophotometer
(Japan) with excitation at 340 and 380 nm and emission at 510
nm, which was performed in a water-jacketed cuvette at 37
°C. Basal and agent-induced changes in the excitation ratio
of fura-2 (340/380 nm) were measured for approximately 10
min after the addition of 50 µmol/mL KCl, 5 µg/mL
isoproterenol, 200 µg/mL LPS and 1 or 2 nmol/mL neutral
sulfate berberine. Maximum (Rmax) and minimum
(Rmin) fluorescence values were determined by adding 50 µL of Triton
X-100 (10%) and 100 µmol/Lethyleneglycol-bis(b-aminoethyl
ether)-N,N,NĄŻ,NĄŻ-tetraacetic acid (EGTA), respectively. The
cardiomyocyte [Ca2+]i was then calculated. In some
experiments, the neonatal rat cardiomyocytes were treated
with 200 mg/mL LPS and/or 2 nmol/mL neutral sulfate
berberine for 1_2 h, after which the cardiomyocyte
[Ca2+]i was examined using fura-2/AM.
Perfused heart preparation Based on a method
described by Stamm et al[10], isolated rat hearts were perfused
in Langendorff mode. In brief, male Sprague-Dawley rats
weighing 200±20 g, obtained from the Guangdong Province
Center for Laboratory Animals were anticoagulated with
heparin (200 U, iv) and anesthetized with intraperitoneal
administration of sodium pentobarbital (35 mg/kg). The hearts
were rapidly excised and arrested in chilled normal saline.
After the aorta was cannulated, hearts were perfused
retrogradely on a Langendorff apparatus with filtered
Krebs-Henseleit buffer at 37 °C and a constant pressure of 80
cmH2O. The Krebs-Henseleit solution was composed of
(mmol/L): NaCl 118, KCl 4.7,
KH2PO4 1.2, NaHCO3
25, CaCl2 2.5, MgSO4 1.2, and glucose 11. The solution was
equilibrated with a 5% CO2 and 95%
O2 gas mixture (pH 7.35_7.45). A small latex balloon filled with fluid was
inserted in the left ventricle and connected to a pressure
transducer, then the maximal rate of left ventricular
pressure rise and fall
(±dp/dtmax) and heart rate were measured
by using a Biolab-410 physiological function system (China)
throughout the experiment. The hearts were divided into 4
groups: the hearts in the control group were perfused with
Krebs-Henseleit buffer; the hearts in the LPS group were
perfused with 100 µg/mL LPS for 20 min followed by
Krebs-Henseleit buffer; in the neutral sulfate berberine plus LPS
group, the hearts were infused with 1 µmol/L berberine for 10
min followed by 100 mg/mL LPS for 20 min, and
Krebs-Henseleit buffer to the end of the experiment; and the hearts
in the berberine group were perfused with 1 µmol/L berberine
for 10 min followed by Krebs-Henseleit buffer to the end of
the experiment.
Statistical analysis Results are expressed as mean±SD.
Statistical analysis was performed using the SPSS software
package (version 8.0). Paired t-tests, StudentĄŻs
t-test and one-way ANOVA followed by the Student-Newman-Keuls
test were used to determine statistical significance. The
difference between means was considered statistically signifi
cant when P was less than 0.05.
Results
Influence of LPS and neutral sulfate berberine on TNF-a secretion in neonatal rat cardiomyocytes LPS stimulated
TNF-a release from cardiomyocytes. In the presence of 10%
fetal bovine serum, untreated cardiomyocytes produced a
basal level of 86.6±7.3 pg/mL of TNF-a. Treatment of
cardio-myocytes for 6 h with LPS at concentrations of 1, 5, 10, and
20 mg/mL all caused a significant increase in the amount of
TNF-a released (Figure 1). In contrast, berberine inhibited
LPS-induced TNF-a release from neonatal rat
cardiomyo-cytes in a dose-dependent manner. Berberine at a
concentration of 4 nmol/mL almost completely prevented the
increase in TNF-a release in cardiomyocytes exposed to 10
mg/mL LPS (Table 1).
Effects of neutral sulfate berberine on
[Ca2+]i changes in neonatal rat cardiomyocytes exposed to KCl,
isopro-terenol, and LPS Addition of 50 mmol/L KCl caused an
increase in the [Ca2+]i of cardiomyocytes from 70.61±4.26
nmol/L to 159.63±11.88 nmol/L (n=6,
P<0.01), and this increase was completely suppressed by pretreatment of the
cardiomyocytes with 2 µmol/L berberine (75.26±6.06 nmol/L;
Figure 2). As shown in Table 2, pretreatment of
cardio-myocytes with 2 µmol/L berberine for 10 min did not
suppress the isoproterenol-evoked increase in
[Ca2+]i of cardio-myocytes. LPS at a concentration of 200 µg/mL evoked a
rapid increase in [Ca2+]i of cardiomyocytes 10 min after
stimulation (Figure 3). Berberine treatment alone for 10 min
stimulated an increase in the
[Ca2+]i of cardiomyocytes, but
did not inhibit the LPS-induced rapid elevation of the
[Ca2+]i of cardiomyocytes (Table 3). However,
[Ca2+]i in cardiomyo-cytes decreased markedly when the cells were treated with
LPS (200 µg/mL) for 1 h, and became significantly elevated
2 h after LPS exposure. Both of these effects were reversed
by 2 µmol/L berberine. Berberine treatment for 1 and 2 h
pro-duced an elevation in
[Ca2+]i of cardiomyocytes (Table 4, 5).
Effects of neutral sulfate berberine and LPS on heart
rate and ±dp/dtmax in isolated rat
hearts Infusion with 100 µg/mL LPS for 20 min significantly decreased the heart rate
from 256±56 beats/min to 140±104 beats/min at 120 min in
isolated rat hearts (P<0.05), whereas this effect was not found
in control hearts. Infusion with 1 µmol/L berberine for 10 min
also reduced the heart rate from 289±35 beats/min to 194±38
beats/min at 120 min (P<0.05). Moreover, berberine did not
antagonize the LPS-induced drop in heart rate (from 253±56
beats/min to 160 ± 49 beats/min at 120 min). As shown in
Tables 6 and 7, perfusion of isolated heart with 100 µg/mL
LPS for 20 min resulted in significantly impaired cardiac
performance at 120 min after LPS challenge, and the maximal rate
of left ventricular pressure rise and fall
(±dp/dtmax) decreased
compared with the controls. In contrast,
±dp/dtmax at 120 min of hearts perfused with 1
µ mol/L berberine for 10 min, followed by 100 µg/mL LPS for 20 min was higher than that
for hearts in the LPS group.
Discussion
The mechanism of LPS-induced cardiodepression
remains controversial. Recently, some evidence has
demonstrated that TNF-a contributes to LPS-induced cardiac
dysfunction, although other mediators have been involved
in this process[10,13]. Kapadia et al
reported that during endotoxemia, TNF-a gene and protein expression increased
in feline myocardium[14]. Furthermore, LPS has been found
to induce the secretion of TNF-a from cultured adult rat
cardiomyocytes via the CD14 signal pathway, LPS has also
been shown to trigger apoptosis in rat cardiomyocytes, which
can be blocked by pretreatment with the soluble
TNF-a fragment, indicating that the cardiodepressant action of LPS
may be in part due to TNF-a-induced apoptosis, which
decreases the number of working myocardial
cells[15]. Inhibition of myocardial
NF-kB activation in transgenic mice constitutively overexpressing a nondegradable
I-kBa in cardiomyocytes can inhibit cardiac TNF-a synthesis and
prevent cardiac dysfunction induced by
LPS[4]. In the present study, we confirmed that LPS stimulated
TNF-a secretion from neonatal rat cardiomyocytes and, importantly, we also
found that neutral sulfate berberine dose-dependently
inhibited TNF-a release from cardiomyocytes exposed to LPS,
although the mechanism by which neutral sulfate berberine
suppresses LPS-stimulated TNF-a release from
cardio-myocytes remains to be further investigated.
There is some evidence that abnormal calcium cycling in
cardiomyocytes may be related to LPS-induced cardiac
depression, although reports from different laboratories are
inconsistent[16_18]. Stamm and colleagues demonstrated that
recirculating perfusion of isolated rat heart with LPS for 30
min significantly impaired myocardial contractility, which was
associated with lower
[Ca2+]i levels and attenuated systolic
increases in [Ca2+]i. They found that these LPS effects were
not only mimicked by rat TNF-a but also blocked by
anti-TNF-a antibody, suggesting a direct relationship between
LPS-induced TNF-a release, abnormal calcium cycling and
reduced contractility in intact
hearts[10]. Thompson et al reported that 18-h exposure to LPS stimulated myocardial
calcium overload and that dantrolene inhibited calcium over
load and improved LPS-induced cardiac
dysfunction[19]. Therefore, decreases in
[Ca2+] i in the early stage and calcium
overload in the later stage after LPS challenge played
important roles in LPS-induced myocardial depression. In the
present study, we observed that neutral sulfate berberine
blocked a KCl-evoked increase in
[Ca2+]i, although neutral sulfate berberine itself induced a rapid rise in
[Ca2+]i in cardiomyocytes. However, neutral sulfate berberine could
not inhibit isoproterenol- and LPS-evoked rapid increases in
cardiomyocyte [Ca2+]i. These data indicated that
LPS-stimulated rapid increases in
[Ca2+]i might involve multiple
mechanisms, including calcium release from the
sarcoplasmic reticulum. However,
[Ca2+]i in cardiomyocytes decreased
markedly when the cells were treated with LPS for 1 h, and
elevated significantly 2 h after LPS exposure, both of which
were reversed by neutral sulfate berberine.
In summary, we observed that neutral sulfate berberine
not only inhibited TNF-a release, but also prevented
abnormal calcium cycling in cardiomyocytes, suggesting that
neutral sulfate berberine may improve LPS-induced cardiac
dysfunction. To further test this hypothesis, we also
examined the effect of neutral sulfate berberine on LPS-induced
myocardial depression in isolated intact rat hearts. We found
that neutral sulfate berberine itself had a negative effect on
frequency in isolated whole rat hearts, and did not reverse
the LPS-induced negative impact on frequency, but
prevented LPS-induced systolic and diastolic ventricular
dysfunction. These findings demonstrated that neutral
sulfate berberine inhibited LPS-stimulated TNF-a release and
normalized intracellular calcium levels in cardiomyocytes
exposed to LPS, and attenuated LPS-induced myocardial
depression. Neutral sulfate berberine may be a potential
therapeutic agent for the treatment of LPS-induced heart
dysfunction, and its in vivo effect on heart function during
endotoxemia deserves to be further investigated.
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