BMC Complementary and

Alternative Medicine BioMed Central

Research article Open Access

Effect of Curcuma longa and Ocimum sanctum on myocardial
apoptosis in experimentally induced myocardial
ischemic-reperfusion injury
Ipseeta Mohanty*†, Dharamvir Singh Arya† and Suresh Kumar Gupta†

Address: Department of Pharmacology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi-110029, India
Email: Ipseeta Mohanty* - [email protected]; Dharamvir Singh Arya - [email protected];
Suresh Kumar Gupta - [email protected]
* Corresponding author †Equal contributors

Published: 19 February 2006 Received: 17 May 2005

Accepted: 19 February 2006
BMC Complementary and Alternative Medicine 2006, 6:3 doi:10.1186/1472-6882-6-3
This article is available from: http://www.biomedcentral.com/1472-6882/6/3
© 2006 Mohanty et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract
Background: In the present investigation, the effect of Curcuma longa (Cl) and Ocimum sanctum
(Os) on myocardial apoptosis and cardiac function was studied in an ischemia and reperfusion (I-
R) model of myocardial injury.
Methods: Wistar albino rats were divided into four groups and orally fed saline once daily (sham,
control IR) or Cl (100 mg/kg; Cl-IR) or Os (75 mg/kg; Os-IR) respectively for 1 month. On the 31st
day, in the rats of the control IR, Cl-IR and Os-IR groups LAD occlusion was undertaken for 45
min, and reperfusion was allowed for 1 h. The hemodynamic parameters{mean arterial pressure
(MAP), heart rate (HR), left ventricular end-diastolic pressure (LVEDP), left ventricular peak
positive (+) LVdP/dt (rate of pressure development) and negative (-) LVdP/dt (rate of pressure
decline)} were monitored at pre-set points throughout the experimental duration and
subsequently, the animals were sacrificed for immunohistopathological (Bax, Bcl-2 protein
expression & TUNEL positivity) and histopathological studies.
Results: Chronic treatment with Cl significantly reduced TUNEL positivity (p < 0.05), Bax protein
(p < 0.001) and upregulated Bcl-2 (p < 0.001) expression in comparison to control IR group. In
addition, Cl demonstrated mitigating effects on several myocardial injury induced hemodynamic
{(+)LVdP/dt, (-) LVdP/dt & LVEDP} and histopathological perturbations. Chronic Os treatment
resulted in modest modulation of the hemodynamic alterations (MAP, LVEDP) but failed to
demonstrate any significant antiapoptotic effects and prevent the histopathological alterations as
compared to control IR group.
Conclusion: In the present study, significant cardioprotection and functional recovery
demonstrated by Cl may be attributed to its anti-apoptotic property. In contrast to Os, Cl may
attenuate cell death due to apoptosis and prevent the impairment of cardiac performance.

Background est. It accounts for a great proportion of cell loss

Recently, the recognition of a different cell death phe- associated with myocardial infarction (MI) and / or
nomenon 'Apoptosis' has become a major clinical inter- ischemia-reperfusion (IR). Cell loss through apoptosis

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contributes to the impairment of cardiac performance and tective action of these herbal extracts was confirmed by
also plays an important role in myocardial remodeling assessing the severity of pathological changes.
processes [1,2]. Induction of apoptosis is implicated in
myocardial I-R injury among other cardiovascular dis- Methods
eases [3,4]. Various studies have demonstrated that not Experimental animals
only reactive oxygen species (ROS) per se, but also their Adult male Wistar rats, 10 to 12 weeks old, weighing 150
oxidation products and other secondary messenger mole- to 200 g were used in the study. The study protocol was
cules generated by ROS can trigger the programmed cell reviewed and approved by the Institutional Animal Ethics
death [5]. It has been reported that these programmed cell Committee and conforms to the Indian National Science
death pathways can be inhibited by antioxidants [6,7] Academy Guidelines for the Use and Care of Experimental
However, there are few studies addressing the inhibition Animals in Research. Animals were obtained from the
of apoptosis and it's directs on myocardial contractility. Central Animal Facility of All India Institute of Medical
Since apoptosis, is a genetically regulated process, hence, Sciences, New Delhi, India and were maintained under
a better understanding of the cellular mechanisms that standard laboratory conditions in the department animal
control apoptosis, could lead to defining novel and effec- house. Rats were housed in polyacrylic cages (38 × 23 × 10
tive therapeutic strategies to limit the amount of tissue cm) with not more than four animals per cage. They were
damage in patients with MI [2-4]. housed in an air-conditioned room and were kept in
standard laboratory conditions under natural light and
Curcuma longa (Cl), common Indian dietary pigment and dark cycles (approximately 14 h light/10 h dark) and
spice has been shown to possess a wide range of therapeu- maintained at humidity 60 ± 5% and an ambient temper-
tic utilities in the traditional Indian medicine. It's role in ature of 25 ± 2°C. All experiments were performed
wound healing, urinary tract infections, liver ailments are between 9.0 and 16.0 h. The animals were allowed free
well-documented [8]. The active component of turmeric excess to standard diet (Ashirwad; Chandigarh) and tap
identified as curcumin exhibits a variety of pharmacolog- water ad libitum and allowed to acclimatize for one week
ical effects including antioxidant, adaptogenic, anti- before the experiments. Commercial pellet diet contained
inflammatory and anti-infectious activities [9,10]. Oci- 24% protein, 5% fat, 4% fiber, 55% carbohydrates, 0.6%
mum sanctum (Os), commonly known, as Tulsi in India is calcium, 0.3% phosphorous, 10% moisture and 9% ash
a local herb containing potent antioxidants flavanoids w/w.
(orientin, vicenin) and phenolic compounds (eugenol,
cirsilineol, apigenin) [11]. The ancient systems of medi- Chemicals
cine including Ayurveda, Greek, Roman, Siddha and All Chemicals were of analytical grade, purchased from
Unani, have mentioned its therapeutic applications in car- Sigma Chemical Co., St Louis, USA. Hydro-alcoholic
diovascular disorders, diabetes and asthma [12,13]. How- lyophilized extract of Ocimum sanctum was procured from
ever, only few studies are presently available that Dabur Research Foundation, and aqueous extract of Cur-
documents its cardioprotective potential. Therefore with cuma longa was obtained from Sanat Research Laborato-
the point of view that it might be interesting and possibly ries, India. The ABC staining kit and primary (Bax mouse
fruitful to study the anti-apoptotic properties of Curcuma monoclonal IgG2b and Bcl-2 mouse monoclonal IgGI) &
longa and Ocimum sanctum (medicinal herbs widely used secondary antibodies (Anti mouse IgG) were procured
for the treatment of various diseases in Ayurveda, the from Santa Cruz Biotechnology, USA. TUNEL assay kit
Indian System of Medicine) and their effect on ventricular was purchased from Roche Diagnostics, USA. Double dis-
function, the present investigation was planned to unravel tilled water was used in all biochemical assays.
the molecular mechanism of the cardioprotective poten-
tial of these time tested herbal plants [8-13]. Treatment protocol
The animals were randomly divided into four main
The relative involvement of apoptosis in cardiac I-R was groups comprising of thirteen animals each.
evaluated and the anti-apoptotic activity of these herbs
was investigated using immunohistochemical localiza- Group 1 – Saline control group – Sham group
tion of Bax and Bcl-2 proteins and TUNEL staining. To Rats were administered 0.9% normal saline for a month
correlate the apoptotic cell death and altered cardiac per- and then sacrificed on the 31st day. The animals were sub-
formance during myocardial I-R changes in the hemody- jected to the entire surgical procedure and thread was
namic variables were also monitored in the present study. passed beneath the coronary artery but the LAD coronary
Alterations in MAP, HR, LVEDP, (+) & (-)LVdP/dt were artery was not ligated.
monitored and recorded at preset time points throughout
the experimental period (1 hour 45 minutes). Cardiopro-

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Pilot study for measurement of MAP and HR. The left jugular vein
Cl at the doses of 25, 50, 100 & 200 mg/kg and Os at the was cannulated with polyethylene tube for continuos
doses of 25, 75 & 150 mg/kg were screened in the murine infusion of 0.9% saline solution. A left thoractomy was
model of isoproterenol induced myocardial necrosis and performed at the fifth intercostal space and the pericar-
the optimum dose exhibiting maximum cardioprotective dium was opened to expose the heart. The left anterior
effect was evaluated. Cl (100 mg/kg) and Os (75 mg/kg) descending coronary artery (LAD) was ligated 4–5 mm
doses respectively were found to be the most effective in from its origin by a 5-0 silk suture with a atraumatic nee-
functional recovery of the heart and favorable restoration dle and ends of this ligature were passed through a small
of biochemical and histopathological alterations. Hence, vinyl tube to form a snare. After the completion of the sur-
these doses were selected for further evaluation singularly gical procedure, the heart was returned to its normal posi-
as well as in combination in the ischemia and reperfusion tion in the thorax. The thoracic cavity was covered with
model of myocardial injury. saline-soaked gauze to prevent the heart from drying. The
animals were then allowed to stabilize for 15 min before
Group 2 – Ischemia and reperfusion group – Control IR LAD ligation. Myocardial ischemia was induced by one
In this group, healthy experimental rats were adminis- stage occlusion of the LAD by pressing the polyethylene
tered 0.9% normal saline for 30 days; thereafter, on the tubing against the ventricular wall and then fixing it in
31st day, the experimental animals were subjected to 45 place by clamping the vinyl tube with a hemostat. A wide
min LAD coronary artery ligation and 60 min reperfusion bore (1.5 mm) sterile metal cannula was inserted into the
induced myocardial injury. cavity of the left ventricle from the posterior apical region
of the heart. The cannula was connected to a pressure
Group 3 – Curcuma longa treated group – Cl-IR transducer (Gould Stathum P231D) and the whole sys-
Aqueous extract of Curcuma longa (100 mg/kg) was tem was filled with heparinized saline (heparin 50 units/
administered orally to healthy experimental animals for 1 ml). Left ventricular systolic and LVEDP was measured on
month. On the 31st day, rats were subjected to 45 min a multichannel polygraph (Grass 7D, USA) from the left
LAD ligation and 60 min reperfusion. The number of ani- ventricular pressures curve at lower and higher sensitivity
mals studied in this group was eleven. of the preamplifier respectively. The maximal rate of rise
and fall of left ventricular pressure {peak (+) LVdP/dt and
Group 4 – Ocimum sanctum treated group – Os-IR peak (-) LVdP/dt} were measured by the electronic differ-
Hydroalcoholic extract of Ocimum sanctum (75 mg/kg) entiator from the signal output of the channel recording
was administered orally to healthy experimental animals left ventricular pressure. A bolus of heparin (30 IU) was
for 30 days, thereafter, on the 31st day the rats were sub- administered immediately before coronary artery occlu-
jected to a protocol of 45 min LAD ligation and 60 min sion for prophylaxis against thrombus formation around
reperfusion induced myocardial injury. The number of the snare. The animals then underwent 45 min of
animals studied in this group was thirteen. ischemia, confirmed visually in situ by the appearance of
regional epicardial cynosis and ST-segment elevation. The
The detailed experimental protocol used in the present myocardium was reperfused by releasing the snare gently
study is as follows: for a period of 60 min. Successful reperfusion was con-
firmed by visualization of arterial blood flow through the
1) Surgical procedure: infarction protocol and artery, appearance of hyperemia over the surface of the
hemodynamic studies previously ischemia cynotic segment. At the end of reper-
Rats of all the experimental groups were anesthetized fusion period, animals were sacrificed for immunohisto-
intraperitoneally with pentobarbitone sodium (60 mg/ chemical and histological studies by an overdose of
kg). Atropine was co-administered with the anesthetic to anesthesia.
keep the heart rate elevated especially during the surgery
protocol and reduce broncho-tracheal secretions. The 2) Apoptotic studies
body temperature was monitored and maintained at i) Immunostaining for the localization of Bax and Bcl-2 proteins
37°C throughout the experimental protocol. The neck A monoclonal mouse anti-human Bcl-2 and Bax proteins
was opened with a ventral midline incision, and a trache- as the primary antibody were used for Bcl-2/Bax immuno-
ostomy was performed and the rats were ventilated with histochemical staining. The ImmunoCruz Staining Sys-
room air from a positive pressure ventilator (Inco, India) tems utilizes a horseradish peroxidase (HRP)-streptavidin
using compressed air at a rate of 70 strokes/min and a complex for staining of formalin-fixed paraffin-embedded
tidal volume of 10 ml/kg. The right carotid artery was can- myocardial sections. Indirect immunoperoxidase staining
nulated and the cannula filled with heparinized saline was performed as described with some modifications
was connected to the cardiac output monitor CARDIOSYS [27]. Briefly, 4–6 micron thick fixed paraffin tissue sec-
CO-101 (Experimetria, Hungary) via a pressure transducer tions were subjected to the following immunohistochem-

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Table 1: Changes in Bax, Bcl-2 protein expression and TUNEL positivity in the different experimental groups

Bax (%) Bcl-2 (%) TUNEL (%)

Sham 3.5% ± 0.4 1.86 ± 0.17 0.2 ± 0.01

Control IR 9.80 ± 0.50### 1.45 ± 0.21 3.0 ± 0.20###
Cl-IR 4.4 ± 0.28** 7.7 + 0.62*** 0.8 ± 0.04*
Os-IR 6.4 ± 0.62* 1.80 ± 0.07 2.4 ± 0.09

Each value is expressed as Mean ± SD of eight experiments. ###p < 0.001 Vs Sham; *p < 0.05, **p < 0.01, ***p < 0.0001 Vs Control IR. Total cell
counts and Bax, Bcl-2 and TUNEL positive cells in the specimens were determined by means of a light microscope. The Bax, Bcl-2 and TUNEL
positive cells were expressed as percentage of normal nuclei.

ical procedure for the localization of Bax and Bcl-2 Score (-): Absence of any inflammation, edema and
proteins using specific mouse monoclonal primary anti- necrosis
bodies. Sections are first blocked, and then incubated in
primary antibody. Biotinylated secondary antibody is Score (+): Focal areas of inflammation, edema and necro-
added followed by the addition of HRP-Straptavidin com- sis
plex. The target protein (Bax/Bcl-2) was visualized by
incubation in peroxidase substrate (H2O2) using DAB Score (++): Patchy areas of inflammation, edema and
(3,3' diaminobenzidine) as the chromogen. necrosis

ii) Terminal Deoxyribonucleotidyl Transferase-Mediated dUTP Nick Score (+++): Confluent areas of inflammation, edema and
End Labeling (TUNEL Assay) necrosis
Myocardial apoptosis was quantified by detection of DNA
fragmentation using the TUNEL technique. Briefly, the Score (++++): Massive areas of inflammation, edema and
enzyme terminal deoxynucleotidyl tranferase was used to necrosis
incorporate residues of digoxigenin nucleotide into 3' OH
ends of DNA fragments. The free end of cellular DNA was Statistical analysis
labeled by incubating the specimens in streptavidin con- All numerical data in text, figures and tables are expressed
jugated to horse radish peroxidase enzyme and peroxidase as the mean ± SD. Statistical analysis was performed by
substrate. The signal of terminal deoxynucleotidyl tran- one-way analysis of variance (ANOVA) or repeated meas-
ferase-mediated dUTP nick end labeling (TUNEL) assay ures ANOVA when data were compared at different time
was used to identify apoptotic cells using secondary reac- points within a study group and for time courses between
tion with antibodies and DAB chromogen [25]. The slides study groups, followed by the Bonferroni post hoc test.
were counterstained in hematoxylin and total cell counts Differences were considered statistically significant at p <
and TUNEL positive cells in the specimens were deter- 0.05.
mined by means of a light microscope. The cells with clear
nuclear labeling were defined as TUNEL positive cells. The Results
apoptotic cells i.e. TUNEL positive cells were expressed as Effects of Curcuma longa (Cl) and Ocimum sanctum
percentage of normal nuclei. (Os) on
1) Hemodynamic variables following I-R induced myocardial injury
3) Histopathological studies i) Mean Arterial Pressure (MAP) and Heart Rate (HR)
At the end of the experiment, myocardial tissue was The initial value of MAP and HR in the control IR group
immediately fixed in 10% buffered neutral formalin solu- was 124.7 ± 9.4 mm Hg (Graph 1) and 349 ± 22.6 beats/
tion. The tissues were carefully embedded in molten par- min (Graph 2) respectively. Both the hemodynamic vari-
affin with the help of metallic blocks, covered with ables in the control IR group remained depressed
flexible plastic moulds and kept under freezing plates to throughout the I-R duration as compared to sham base-
allow the paraffin to solidify. Cross sections (5 µm thick) line values.
of the fixed myocardial tissues were cut. These sections
were stained with hematoxylin and eosin (H&E) and vis- In the Cl treated group the baseline values of MAP (Graph
ualized under light microscope to study the light micro- 1) and HR (Graph 2) were 126.2 ± 13.9 mm Hg and 339
scopic architecture of the myocardium. The investigators ± 35.3 beats/min respectively. Cl treatment failed to sig-
performing the histologic evaluation were blind to bio- nificantly restore both the hemodynamic parameters as
chemical and hemodynamic results and to treatment allo- compared to control IR values at similar time points. The
cation. The degree of necrosis was graded and scored as value of MAP and HR remained more or less in the same
follows: range as observed in the control IR group, during the

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Figure 2longa
Curcuma
expression of Bax
(100protein
mg/kg)versus
significantly
the control
attenuated
IR group
the
Curcuma longa (100 mg/kg) significantly attenuated the
expression of Bax protein versus the control IR group.

Figure for
Representative
stained 1 Bax photomicrographs
protein of ventricular tissue 1). During the entire ischemic duration, the value of MAP
Representative photomicrographs of ventricular tissue was more or less comparable to control IR recording at
stained for Bax protein. Ischemia and reperfusion induced
similar time points. The MAP showed a gradual decline
myocardial injury significantly increased the expression of
Bax protein compared with non-ischemic tissue. Figures are with the progression of the reperfusion period. However,
representative of 5 separate experiments. at 60 min following reperfusion a significant (p < 0.05)
correction in the value of this parameter was recorded in
the Os treated group as compared to control IR group. The
entire period of LAD coronary artery ligation and reper- initial value of HR in the Os treated group was 352 ± 25.1
fusion as compared to control IR group. beats/min (Graph 2). Os treatment did not bring about
any significant change in HR when compared to control
The MAP recording in the Os treated group, before LAD IR values during the entire duration of 45 min of ischemia
coronary artery ligation, was 123 ± 11.9 mm Hg (Graph and 60 min of reperfusion.

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ii) Left ventricular (+) and (-) maximal rate of change of pressure {(+)
& (-) LVdP/dt}
In the control IR group, the (+) LVdP/dt and (-) LVdP/dt
values before LAD occlusion was 3140 ± 357 mm Hg/s
(Graph 3) and 3004 ± 323 mm Hg/s (Graph 4) respec-
tively. At 5 and 45 min post occlusion there was a signifi-
cant (p < 0.05) fall in (+)LVdP/dt in the control IR group
and reperfusion after 45 min of ischemia also brought
about a significant fall in (+) LVdP/dt at 15, 30, 45 (p <
0.05) & 60 (p < 0.001) min compared to sham baseline
values. (-)LVdP/dt in the control IR group remained sig-
nificantly depressed throughout the entire ischemic and
reperfusion duration, the maximal fall was recorded at the
end of the reperfusion period (p < 0.001) as compared to
sham.

The baseline values of (+) & (-) LVdP/dt in the Cl treated

group were 3202 ± 157 and 3142 ± 257 mm Hg/s respec-
tively (Graph. 3 & 4). Although there was a steady and
continuous fall in the (+) & (-) LVdP/dt in this group dur-
ing the experimental duration, the value of both these
parameters were higher throughout the 45 min of LAD
occlusion and 1 h reperfusion period as compared to the
control IR group. At 60 min post reperfusion, Cl signifi-
cantly improved myocardial contractility (p < 0.01) and
relaxation (p < 0.05) in comparison to control IR values at
same time points.

The baseline values of (+) & (-) LVdP/dt in the Os treated

group before LAD coronary artery occlusion was 3184 ±
125 and 2953 ± 165 mm Hg/s respectively (Graph 3 & 4).
Os treatment failed to significantly improve both myocar-
dial contractility and relaxation during ischemia and
reperfusion duration as compared to control IR values at
similar time points.

iii) Left Ventricular End Diastolic Pressure (LVEDP)

In the control IR group, following LAD coronary artery
occlusion, the LVEDP started rising gradually from its
baseline value of 3.3 + 0.91 mm Hg and it remained ele-
vated throughout the ischemic duration as compared to
sham (Graph 5). The increase in this variable was most Figure
icant
control
Ocimumalteration
IR
3sanctum
groupin(75
themg/kg)
expression
treatment
of Baxdidprotein
not result
versus
in signif-
the
Ocimum sanctum (75 mg/kg) treatment did not result in signif-
significant (p < 0.001) at 45 min of ischemia. On reper-
icant alteration in the expression of Bax protein versus the
fusion, there was a marked and sudden fall in the value of control IR group.
this parameter during the reperfusion phase, but it
remained significantly increased at 5 (p < 0.001) and 15
(p < 0.01) min post reperfusion compared to the sham
group.
group was comparable to control IR recordings at the sim-
The baseline value of LVEDP in the Cl treated group was ilar time points.
3.4 ± 0.25 mm Hg (Fig 5). Cl treatment significantly cor-
rected the elevated LVEDP levels at 15 min (p < 0.001), 25 The baseline value of LVEDP in the Os treated group was
min (p < 0.01), 35 min (p < 0.05) and 45 min (p < 0.001) 3.8 + 0.64 mm Hg (Graph 5). After 5 min of coronary
as compared to control IR values. Thereafter during the artery occlusion LVEDP increased significantly and
reperfusion duration the value of LVEDP in the Cl treated remained elevated during LAD occlusion period in com-

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Figure for
Representative
stained 4 Bcl-2photomicrographs
protein of ventricular tissue
Representative photomicrographs of ventricular tissue
stained for Bcl-2 protein. Coronary occlusion and reper-
Figure
Curcuma
cated
control
byIR
5dark
longa
group
brown
(100 mg/kg)
positive
upregulated
immunoreactivity
Bcl-2 protein
compared
as indi-
to
fusion resulted in a slight reduction (non significant) in Bcl-2
Curcuma longa (100 mg/kg) upregulated Bcl-2 protein as indi-
expression in the control IR group compared to non-
cated by dark brown positive immunoreactivity compared to
ischemic tissue. Figures are representative of 5 separate
control IR group.
experiments.

parison to sham. However, in the Os treatment group the

value of LVEDP was significantly lower at 15 and 45 min increased the expression of Bax protein (p < 0.001) com-
of ischemia (p < 0.01) as compared to control IR values at pared with non-ischemic tissue from 3.50 ± 0.40 to 9.80
the same time points. Post reperfusion the LVEDP values ± 0.50% (Fig 1). Bax expression was significantly attenu-
in the Os treated group were comparable to control IR val- ated to 4.4 ± 0.28% and 6.4 ± 0.62% respectively in the Cl-
ues. IR (p < 0.01, Fig 2) and Os-IR (p < 0.05, Fig. 3) treated
groups as compared to control IR.
2) Myocardial apoptotic parameters following I-R induced myocardial
injury ii) Myocyte Bcl-2 protein expression
i) Myocyte Bax protein expression Bcl-2 protein was expressed in the sham myocardium as
Slight Bax immunoreactivity (3.5% ± 0.4%) was observed indicated by slight positive Bcl-2 immunoreactivity in the
in the myocytes of the sham group (Table 1). Ischemia myocytes. The basal expression of Bcl-2 was found to be
and reperfusion induced myocardial injury significantly 1.86 ± 0.17% (Table 1). Coronary occlusion and reper-

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Figure
Ocimum
min
any
sus the
significant
of ischemia
control
6sanctum
change
IR
and
(75
group
1mg/kg)
in
h of
thereperfusion
treated
expression
group
failed
of Bcl-2
subjected
to demonstrate
protein
to 45
ver-
Ocimum sanctum (75 mg/kg) treated group subjected to 45
min of ischemia and 1 h of reperfusion failed to demonstrate
any significant change in the expression of Bcl-2 protein ver-
sus the control IR group.
Figure for
Representative
stained 7 nick-end
photomicrographs
labeling (TUNEL)
of ventricular
for DNA breaks
tissue
Representative photomicrographs of ventricular tissue
stained for nick-end labeling (TUNEL) for DNA breaks. In
the Control IR group a large number of TUNEL positive cells
are observed subsequent to ischemia and reperfusion injury.
Figures are representative of atleast 5 separate experiments.
fusion resulted in a slight reduction (non-significant) in
Bcl-2 expression compared with non-ischemic tissue (Fig.
4). Significant upregulation in the expression of Bcl-2 was number of TUNEL positive cells expressed as percentage
observed in the Cl (p < 0.001) treated group in compari- of total normal nuclei was significantly increased subse-
son to control IR (Fig. 5). However, in the Os (75 mg/kg, quent to ischemia and reperfusion induced myocardial
Fig. 6) group there was no significant change in the injury in the control IR group (3.0 ± 0.2%, p < 0.001, Fig.
expression of Bcl-2 protein and it was found to be compa- 7) compared to sham non-ischemic myocardium as indi-
rable to control IR group i.e. 1.80 ± 0.07%. cated by increased intensity of TUNEL staining. The
TUNEL positivity was significantly attenuated to 0.8 ±
iii) TUNEL positivity 0.04% in the Cl-IR (p < 0.05, Fig. 8) group as compared to
TUNEL positivity was expressed as percentage of total nor- control IR. However, Os (75 mg/kg, Fig. 9) treated group
mal nuclei. Slight TUNEL positive staining was detected in did not demonstrate any significant anti-apoptotic activ-
the sham group (0.2% ± 0.01%, Table 1). However, the ity in comparison to control IR group. The percentage of

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Figure
Ocimum
min
decrease
of ischemia
9sanctum
TUNELand
(75
positivity
1mg/kg)
h of reperfusion
astreated
compared
group
failed
tosubjected
control
to significantly
IRtogroup
45
Ocimum sanctum (75 mg/kg) treated group subjected to 45
Figure
Relative
tive
cuma cells
longa
8to
was
(100
thesignificantly
control
mg/kg) IR decreased
group the by
number
treatment
of TUNEL
with Cur-
posi- min of ischemia and 1 h of reperfusion failed to significantly
Relative to the control IR group the number of TUNEL posi- decrease TUNEL positivity as compared to control IR group.
tive cells was significantly decreased by treatment with Cur-
cuma longa (100 mg/kg).
tion of inflammatory cells and edema as compared to the
control IR group. However, Os (75 mg/kg) treatment
TUNEL positive cells in the Os-IR was found to be 2.4 ± failed to preserve the myocardial cellular integrity as evi-
0.09%. denced by patchy areas of myonecrosis, edema and mod-
erate degree of inflammation in this group, which was
3) Myocardial histology and histopathology following I-R induced comparable to that observed in control IR group (Table
myocardial injury 2).
Microscopic histology revealed that the non-infarcted
myocardium in the sham group was characterized by an Discussion
organized pattern and shows normal architecture of the Prolonged severe ischemia leads to cardiac cell death.
myocardium (Table 2). Contrastively, on histological Necrosis previously was regarded as the only mode of cell
evaluation, rat hearts, subjected to I-R (Control IR) dem- death, whereas, now there is accumulating evidence that
onstrated marked edema, confluent areas of myonecrosis, in addition to overt necrosis, a subset of cells also die by
myofiber loss and mild inflammation as compared to apoptosis, (the programmed cell death). The relative con-
those in the sham group (Table 2). Cl (100 mg/kg) treated tributions of necrosis and apoptosis to cell death in
rats demonstrated marked structural improvement spe- ischemia and reperfusion are still open to debate,
cially with regard to the degree of myonecrosis, infiltra- although necrosis appears to dominate during ischemia,

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Table 2: Light microscopic changes observed in the different experimental groups

Necrosis Edema Inflammation

Sham - - -
Control IR ++ +++ +
Cl-IR + ++ +
Os-IR ++ ++ +

The relative pathological changes are ranked from - = No change; + = Focal change; ++ = Patchy change; +++ = Confluent change and ++++ =
Massive change.
and apoptosis may dominate during reperfusion. There is group. This observation receives support from earlier
now evidence that apoptosis occurs during sustained studies [18,19]. In addition, consistent with increase in
ischemia and when reperfusion follows shorter periods of apoptotic cell death, post ischemic reperfusion injury also
ischemia [14,15]. resulted in significant depression of left ventricular
dynamics, peripheral hemodynamics (MAP) and HR.
The progressive loss of cardiomyocytes in a heart that is
already compromised leads to further deterioration of car- Cl treatment significantly reduced the percentage of
diac function, conduction disturbances due to degenera- TUNEL positive cells Vs the control IR group, demonstrat-
tion of SA, AV and inter-nodal pathway, cardiac ing its significant anti-apoptotic activity. Treatment with
remodeling and cardiomyopathy [3,4]. The fact that Cl was associated with greater Bcl-2 and attenuated Bax
apoptosis plays a role in the tissue damage seen after myo- expression as compared to the control IR group. Curcu-
cardial infarction has pathological and therapeutic impli- min, the active ingredient of the rhizome of the turmeric
cations. If indeed cardiomyocyte apoptosis plays an plant (Curcuma longa), a commonly used spice, has been
important role in initiation and progression of cardiac reported to prevent cancer in animal tumor models possi-
diseases, drugs that effectively and specifically inhibit bly by its apoptosis-inducing and antiproliferative influ-
apoptosis might be useful therapeutic agents for attenuat- ences [20-22]. However, in the present study, in contrast
ing myocardial injury due to I-R [16]. to earlier reports, marked anti-apoptotic activity of Cl was
observed.
In reperfused ischemic hearts increase in oxidative stress,
and decrease in antioxidant defense has been reported to Previously we reported that cardioprotective effect of Cl
lead to cardiac dysfunction partly due to apoptosis [4,17]. results from the suppression of oxidative stress and corre-
However, whether or not plant derived agents, known to lates with the improved ventricular function [23]. How-
possess antioxidant activity may reduce myocardial apop- ever, till date there are no in vivo studies, which have
tosis induced by I-R, and thus improve ventricular func- explored the relationship between the putative anti-apop-
tion and thus attenuate MI has not been directly totic effects of Cl on the functional recovery of ischemia
investigated. reperfused myocardium. The present study demonstrates
that Cl treatment resulted in preserved left ventricular
In the present study, TUNEL positivity and the immuno- function as reflected by a significant increase in the indices
histochemical localization of Bax, an inducer of apoptosis of contractility (+) LVdP/dt, relaxation (-) LVdP/dt and
and Bcl-2 proteins, inhibitors of apoptosis were studied to decrease in preload (LVEDP). It is speculated that, Cl treat-
delineate the involvement of apoptosis in I-R induced ment may have indirectly restored blood flow in the
injury. In order to correlate whether the inhibition of ischemic regions towards normal as assessed by its effi-
apoptosis has any direct effect on cardiac function, hemo- cacy in improving cardiac performance, especially correct-
dynamic function was also monitored and recorded at ing the ischemia and reperfusion-induced increase in
preset time points throughout the experimental period. LVEDP. However, Cl did not significantly affect MAP and
Cardioprotective activity of these herbs was confirmed by HR. The anti-apoptotic effect of Cl, improved ventricular
assessing the severity of pathological changes. functions with improved histologic features suggests that
treatment with this agent may exert cardioprotective
Results of the present study demonstrated that in the con- effects following coronary ligation and reperfusion.
trol IR group myocardial I-R injury triggered apoptotic cell
death. Increase in TUNEL staining observed in the control Os treatment did not demonstrate any significant anti-
IR group suggests a role of apoptosis in contribution of apoptotic activity as determined by TUNEL staining and
myocardial injury following I-R. A slight reduction in Bcl- immunohistochemical results. No significant change in
2 expression and significant increase in Bax expression as the expression of Bax and Bcl-2 proteins was observed
compared with sham group was observed in the control IR with Os treatment as compared to control IR in the

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present study. It markedly increased MAP, HR and signif- basic physiological and pathological mechanisms rele-
icantly reduced the surrogate preload marker LVEDP as vant to myocardial ischemia and reperfusion injury and
compared to control IR. However, it failed to significantly give new insight to novel therapeutic targets and strategies
improve the left ventricular contractility and relaxation for its treatment. However, the present study provides a
and failed to significantly modulate the histopathologic lead for further exploring other mechanisms contributing
alterations compared to control IR group. Results of the to the cardioprotective effect of Cl. Whether the conclu-
present study demonstrate that Os does not possess signif- sions drawn on the basis of the current data can be extrap-
icant cardioprotective effects. olated to clinical setting, remains to be defined by well-
controlled studies in-patients. Nonetheless, the results of
The relationship between the possible anti-apoptotic the present study are rather encouraging, because they
effects of Cl on the functional recovery of ischemic reper- could unravel a new therapeutic approach for the preven-
fusion injury of the heart has been well elucidated by the tion and/or treatment of ischemic heart disease.
dosing protocol of the present study. The exact mecha-
nism by which Cl may reduce myocardial ischemia and Conclusion
reperfusion induced myocardial apoptosis is far from In the present investigation it was observed that subse-
clear presently. Recently it has been reported that Curcu- quent to ischemia and reperfusion injury, Curcuma longa
min, an active principle of Curcuma longa reduces cardi- treated group demonstrated significant anti-apoptotic
omyocytic apoptosis. Curcumin an inhibitor of NF- property, which might contribute to the observed preser-
kappaB, ameliorated the surge of pro-inflammatory vation in cardiac function and cardioprotective effects.
cytokines during cardiopulmonary bypass (CBP) and Furthermore, the myocardial salvaging effects of Curcuma
decreased the occurrence of cardiomyocytic apoptosis longa were supported by histopathological studies. In con-
after global cardiac ischemia/reperfusion injury. The trast, Ocimum sanctum did not exhibit any significant anti-
authors proposed that by inhibiting NF-kappaB activa- apoptotic effects and cardioprotection.
tion, the up-regulation of cardiac proinflammatory genes
can be ameliorated, and the activation of matrix metallo- Abbreviations
proteinase can be decreased during CPB, thereby lessen- Curcuma longa (Cl), Ocimum sanctum (Os), ischemia and
ing severity of cardiac mechanical dysfunction after global reperfusion (I-R), mean arterial pressure (MAP), heart rate
cardiac ischemia/reperfusion injury [24,25]. However, in (HR), left ventricular end-diastolic pressure (LVEDP), left
the present study, we have used the aqueous extract of Cl ventricular peak positive (+) LVdP/dt (rate of pressure
and have proposed a different mechanism for its antiap- development) and negative (-) LVdP/dt (rate of pressure
optotic effect and correlated it with the ventricular func- decline) and cardiopulmonary bypass (CBP).
tion. Based on the present findings it can be speculated
that Cl may attenuate apoptosis via a number of mecha- Competing interests
nisms: Upregulation of Bcl-2 may result in formation of The author(s) declare that they have no competing inter-
heterodimers with Bax, resulting in no/fewer free Bax pro- ests.
tein available for homodimerization. If Bax homodimers
predominate cell death will occur, but when Bcl-2 and Bax Authors' contributions
heterodimererization prevails cells can survive. Substan- IM carried out the experimental work, participated in the
tial evidence indicates that the mitochondria play a criti- sequence alignment, drafted the manuscript and per-
cal regulatory role in the signal transduction pathway formed the statistical analysis. SKG conceived the study,
leading to apoptosis [4,26]. Loss of contractile cells in the and participated in its design and coordination. DSA par-
heart poses an additional workload on the remaining via- ticipated in the design of the study and helped to draft the
ble myocytes that may be unbearable, resulting in patho- manuscript. All authors read and approved the final man-
logic stimuli and death signals. In the present study, In uscript.
contrast to Os, Cl treatment may have salvaged these myo-
cytes and prevented cell loss induced by apoptosis. His- Additional material
topathologic evaluation further confirms the
cardioprotective potential of such a treatment.
Additional file 1
Graphs
In order to elucidate, the additional mechanisms by Time course of changes in MAP in different groups. Each value is
which Cl may reduce myocardial apoptosis and the expressed as Mean ± SD of eight experiments. *p < 0.05 Vs Control IR
potential clinical implications of such actions, we need to Click here for file
further investigate the relationship of the detrimental [http://www.biomedcentral.com/content/supplementary/1472-
effects of key oxidants and apoptotic signals with reper- 6882-6-3-S1.doc]
fusion injury. This will lead to a better understanding of

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