Introduction
Nicousamide, a novel coumarin-aspirin compound, has
been categorized as a 1.1 class drug and is undergoing stage I
clinical trials in China (1). Its
structure is shown in Fig. 1.
Previous studies have proven its potential to prevent diabetic
nephropathy in rats via the inhibition of advanced glycation end
products (ACEs) (2,3). Besides inhibiting ACEs formation,
nicousamide is a potent inhibitor of the phosphorylation of TGF-β
receptor II (1). By blocking the
TGF-β-Smad signaling pathway, nicousamide retards renal
fibrogenesis and exhibits a renal protective effect.
Recently, we also demonstrated that nicousamide is
able to suppress hypertensive nephropathy in spontaneously
hypertensive rats (SHRs) during the long-term treatment (data under
publication), although its mechanism remains unclear. Nicousamide
shows a marked anti-hypertensive effect in SHRs, while its effect
is weaker compared to losartan. In the present study, we examined
its mechanism on blood pressure (BP) control, by introducing the
two-kidney one-clip (2K1C) model of hypertension in order to
investigate its anti-hypertension action. Additionally, by
analyzing angiotensin II (Ang II), nitric oxide (NO), endothelin-1
(ET-1), nitric oxide synthase (NOS) and calcitonin gene-related
peptide (CGRP) levels in the plasma of the model animals, we aimed
to understand the underlying mechanisms by which nicousamide
reduces BP. Captopril was also included in the study as the
positive control to better evaluate the potency of nicousamide.
Materials and methods
Animals
Six-week-old adult male Wistar rats, weighing
160–180 g, were purchased from the Institute of Laboratory Animal
Science, Chinese Academy of Medical Sciences (Beijing, China). They
were kept in a specific-pathogen-free facility with food and water
ad libitum, at a constant temperature (22±2°C) and a daily
illumination period of 12 h (7:00 a.m.–7:00 p.m.). The study was
approved by our Institutional Animal Care Committee of China, and
the procedures were in accordance with the National Institute of
Health Guide for the Care and Use of Laboratory Animals.
Reagents and drugs
Nicousamide was synthesized by the Department of
Medicinal Chemistry, Institute of Materia Medica, Chinese Academy
of Medical Science (Beijing, China), with 99% purity by high
performance liquid chromatography (HPLC). Its chemical structure is
shown in Fig. 1. Captopril was
purchased from Beijing Taiyang Pharmaceutical Industry Co., Ltd.
(Beijing, China).
Induction of renovascular hypertension,
grouping and treatment
Induction of renal hypertension was carried out in
rats according to the 2K1C model modified by previous studies
(4,5). Briefly, rats were anaesthetized with
3% sodium pentobarbital [30 mg/kg by intraperitoneal injection
(i.p.)]. The left posterior side of the animal was shaved and
sterilized with 70% ethanol. An ∼2 cm incision was made
subcutaneously through the muscles just below the ribs. The left
kidney was then exposed and retracted to expose the renal artery. A
silver clip with an internal diameter of 0.15–0.20 mm was placed
around the left renal artery as close as possible to the aorta. The
clip was then placed with the slit opening facing the abdomen. The
contralateral kidney was left intact. The muscles and skin were
sutured and the rats were allowed to recover from anaesthesia and
left for six weeks to develop hypertension. During this time
period, SBP and the heart rate (HR) were measured at weekly
intervals. Sham-operated rats underwent the same surgical procedure
except for the clip placement. After six weeks, only animals with
SBP >160 mmHg were selected for further drug administration. The
BP and HR was measured by tail-cuff plethysmography (BP-98A;
Softron, Tokyo, Japan) with prior training to minimize variability
in the BP measurement.
The hypertensive rats were randomized into five
groups: the sham and model controls, captopril [6 mg/kg,
periocularly (p.o.)], and nicousamide (30 and 60 mg/kg, p.o.)
groups. Administration continued for three weeks and systolic blood
pressure (SBP), diastolic blood pressure (DBP) and HR were measured
at the end of treatment.
Preparation of blood samples for
biochemical analysis
Subsequent to 3-week nicousamide treatment, blood of
rats was sampled through the eyes after rats were anesthetized with
diethyl ether. Blood plasma was obtained for biochemical analysis.
Blood samples (3 ml) were placed in tubes containing 1% heparin
(0.1 ml). Plasma was obtained by centrifugation at 1200 × g for 20
min (4°C).
Determination of NO level
The total amount of NO (NOx) was indirectly
estimated in terms of its main metabolites, nitrate and nitrite by
the Griess reaction after the sample was de-proteinized with
absolute ethanol. The absorbance was measured at 540 nm and the
results were expressed in the serum samples as μmol/l.
Determination of Ang II, ET-1, NOS and
CGRP levels in rat plasma
The plasma concentration of Ang II was determined by
Ang II radioimmuoassay kits (Beijing North Institute of Biological
Technology, Beijing, China) (6),
and ET-1 level in plasma was measured by a colorimetric
immunometric enzyme immunoassay kit (IBL, Tokyo, Japan).
Determination of the total NOS concentration in plasma was
determined using the ELISA kit (Biomart, Shanghai, China). CGRP was
determined using the ELISA kit (Cayman Chemical, Michigan, USA),
The detailed procedure was carried out following the manufacturer’s
instructions.
Statistical analysis
Data were expressed as the mean ± SEM. A comparison
between the mean values of different groups was carried out using
one-way analysis of variance (ANOVA), followed by the Tukey-Kramer
post hoc test for multiple comparisons. In the data analysis,
P<0.05 was considered to indicate a statistically significant
difference.
Results
Nicousamide decreased the SBP and DBP in
2K1C hypertensive rats
Six weeks after 2K1C operation, the hypertensive
rats (SBP >160 mmHg) were enrolled in the treatment. As shown in
Table I, after three weeks of
treatment, the model group still had a markedly higher average DBP
and SBP compared to the sham group (P>0.05). Simultaneously,
nicousamide treatment reduced the SBP by ∼9% at two doses, compared
to the model control (P<0.05), and similar to captopril,
nicousamide also markedly reduced DBP. However, nicousamide did not
exhibit a statistically significant effect on HR in 2K1C
hypertensive rats.
 | Table I.Values of SBP, DBP and HR after 3-week
administration of nicousamide and captopril. |
Table I.
Values of SBP, DBP and HR after 3-week
administration of nicousamide and captopril.
| Groups | SBP (mmHg) | DBP (mmHg) | HR (beats/min) |
|---|
| Sham control | 154.1±9.14 | 125.7±15.79 | 331.6±44.9 |
| Model control | 172.91±10.17a | 145.7±12.82a | 354.6±29.76 |
| Captopril (6
mg/kg) | 157.8±11.74c | 130.3±15.41b | 355.4±32.74 |
| Nicousamide (30
mg/kg) | 158.3±14.46b | 125.9±12.27c | 362.7±32.51 |
| Nicousamide (60
mg/kg) | 158.1±18.87b | 127.8±26.97 | 336.1±58.38 |
Nicousamide decreased Ang II and ET-1
levels in the plasma of renovascular hypertensive rats
As shown in Table
II, nicousamide treatment decreased the plasma Ang II level in
2K1C rats by 29 and 37%, respectively, at the doses of 30 and 60
mg/kg (P<0.05), compared to the model control. This treatment
therefore had a better effect compared to captopril.
| Table II.Values of Ang II, ET-1, NO, NOS and
CGRP after 3-week administration of nicousamide. |
Table II.
Values of Ang II, ET-1, NO, NOS and
CGRP after 3-week administration of nicousamide.
| Groups | Sham control | Model control | Captopril | Nicousamide (30
mg/kg) | Nicousamide (60
mg/kg) |
|---|
| Ang II (pg/ml) | 80.7±14.9 | 139.3±11.1a | 130.6±53.6 | 99.2±53.2b | 87.4±28.9b |
| ET-1 (pg/ml) | 176±87 | 331±190a | 205±88 | 260±116b | 264±129b |
| NO (μmol/l) | 13.3±9.4 | 5.2±2.4a | 10.2±7.4 | 9.6±5.5 | 13.6±9.6b |
| NOS (U/mg prot) | 26.6±3.0 | 26.1±2.7 | 24.4±3.2 | 20.9±7.5 | 24.3±4.1 |
| CGRP (pg/ml) | 51±31 | 40±20 | 38±25 | 35±30 | 49±27 |
Compared to the model control, plasma ET-1
concentration in nicousamide-treated groups was reduced by ∼21.5%
at 30 and 60 mg/kg, which was less effective compared to
captopril.
Nicousamide increased NO level in plasma,
but had no effect on NOS
As shown in Table
II, nicousamide increased the plasma NO concentration in
renovascular hypertensive rats at two doses. In order to understand
the underlying mechanisms by which nicousamide reduced the NO
level, the total NOS level in the plasma was tested, and no
statistically significant difference was detected in the model
group and the nicousamide-treated groups.
Nicousamide had no effect on plasma CGRP
levels
Due to large standard deviation (SD), no
statistically significant difference was observed in CGRP
concentration among the five experimental groups, however,
additional investigation is required.
Discussion
Previous studies have shown nicousamide to have a
beneficial effect on hypertensive nephropathy in SHRs, correlated
with anti-hypertension and the downregulation of Ang II (data under
publication). To confirm that nicoumisade reduces BP, the 2K1C
renovascular hypertensive rodent model was introduced in the
present study. The 2K1C rat model of arterial hypertension is
well-established and reflects a common condition in a large number
of humans with secondary forms of hypertension, thus, it is a
renovascular Ang II-dependent form of arterial hypertension
(7,8).
After 2K1C operation for six weeks, we detected a
marked increase in the SBP and DBP in all the groups, with the
exception of the sham group, demonstrating the successful
establishment of a renovascular hypertension model. After a
three-week oral administration of nicousamide and captopril, we
further confirmed that nicousamide had a marked anti-hypertensive
effect, on SBP as well as DBP, and its effect was almost identical
to that of captopril. No notable changes were observed in HR in any
of the groups.
To understand the underlying mechanisms of the anti-
hypertensive effect, the plasma concentration in Ang II, NO, ET-1
and CPRP was examined. Ang II was found to be a strong
vasoconstrictor. In the present study, nicousamide treatment
markedly reduced the Ang II level in a dose-dependent manner,
suggesting that nicousamide may reduce the BP via the suppression
of plasma Ang II.
Accumulating evidence suggests that NO plays a
significant role in the regulation of BP and that impaired NO
bioactivity is an important component of hypertension (9). Mice with disruption of the gene for
endothelial NOS (eNOS) exhibit elevated BP levels compared to the
control animals, suggesting a NOS to the link between impaired NO
bioactivity and hypertension (10).
In the current study, the plasma NO level in the 2K1C model rats
was markedly decreased by 60.9% compared to the sham control.
Notably, nicousamide treatment could raise the plasma NO level in
hypertensive rats in a dose-dependent manner, thus, at a dose of 60
mg/kg, the NO level is almost identical to that of the sham
control, thereby explaining its anti-hypertension effect. To
further investigate this mechanism, we examined the total NOS
concentration in the animal plasma, including eNOS and inducible
NOS (iNOS). However, the nicousamide dose did not have a
statistically significant effect on NOS. These results suggest that
nicousamide reduces the plasma NO concentration in hypertensive
rats, although the detailed mechanism remains to be elucidated.
ET-1 is a paracrine or autocrine hormone, which was
characterized as a vasoconstrictor and is overexpressed in the
vasculature and blood in various models of hypertension, including
the 2K1C model (11). In the
present study, treatment using nicousamide downregulates the ET-1
level in the plasma of hypertensive rats, suggesting the presence
of another action mechanisms of anti-hypertension.
We also examined another potent peptide vasodilator,
CGRP. In subtotal nephrectomy (SN) and salt-induced hypertension,
CGRP plays a compensatory role to attenuate the BP increase in the
absence of an increase in the neuronal synthesis and release of
this peptide (12,13). Therefore, the purpose of this study
was to determine whether or not the mechanism of this
anti-hypertensive activity is through enhanced sensitivity of the
vasculature to the dilator actions of this neuropeptide. However,
preliminary results suggest that nicousamide did not markedly
affect the plasma CGRP concentration.
In summary, nicousamide has a beneficially
anti-hypertensive effect in 2K1C rat model, its mechanism possibly
correlated with Ang II and ET-1 suppression, and the increase of
plasma NO concentration. However, the detailed mechanisms requires
further investigation.
Acknowledgements
The authors would like to thank the
Key Project of the National Eleventh-Five Year Research Program of
China, for their support.
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