Ischemic heart disease represents a global burden on health care resources and will be the leading cause of morbidity and mortality in the world by 2030.[1] Acute occlusion of major coronary artery, leading to myocardial infarction , represent a staggering economic burden on health care resources. An improvement in myocardial pump function and decrease in mortality have been demonstrated in several clinical trials employing early reperfusion.[2] Thus the attention is focused to understand the adaptive mechanism that make the myocardium more resistant to sustained ischemia of longer duration and to restore its viability on reperfusion.
Myocardial ischemic preconditioning is a phenomenon by which a brief episode(s) of myocardial ischemia increases the ability of the heart to tolerate a subsequent prolonged period of ischemic injury. Ischemic preconditioning (IP), first demonstrated by Murry in 1986,[3] is the phenomenon in which non-lethal brief episodes of ischemia and reperfusion enhance tolerance of the heart to subsequent lethal or long time ischemia. Repeated short episodes of ischemia and reperfusion have been demonstrated to make myocardium transiently more resistant to deleterious effects of prolonged ischemia.[4][5] The result of IP may be manifest as a marked reduction in infarct size, myocardial stunning, or incidence of cardiac arrhythmias.[6,7] Pharmacological interventions such as adenosine,[8,9] bradykinin,[10] angiotensin, [11] and opioids receptors [12,13] have conferred ischemic preconditioning like cardioprotective effect. The receptor like bradykinin coupled with signal transduction pathway that inhibit the mPTP (mitochondrial permeability transition pore) during the reperfusion phase following the infarction and the opening of these mPTP completely disrupts mitochondrial function and this lead to cell death either by apoptosis or necrosis.[14]
Literature review
Role of KATP channels in ischemic preconditioning
KATP channels have been reported to play a key role in ischemic preconditioning. Opening of KATP channels provide cardioprotection and inhibition of these channels increase the extent of ischemia and reperfusion-induced myocardial injury.[7] The KATP channels present in a cell are sarcolemmal channels (sarc KATP channel) and an inner mitochondrial membrane channel (mito KATP channel).[15] Both sarcolemmal and the more nebulous mitochondrial KATP channels in the cardiovascular system might have a important role in modulating cardiac function, particularly under conditions of metabolic stress, such as hypoxia, ischemia, and metabolic inhibition when intracellular ATP stores are reduced.[16] The opening of mito KATP channel as a result of ischemic preconditioning have demonstrated to produce cardioprotective effect that may be due to the opening of surface KATP channels during ischemia that was may be facilitated by the activation of IP signalling pathways.[17]
Role of adenosine
Ischemic preconditioning has been reported to produce cardioprotective effect through the release of adenosine. Adenosine mediates its cardiovascular actions via four known receptor subtypes (A1, A2A, A2B and A3). All these receptors are expressed in different cell types of the heart and blood vessels, but A1A receptors (A1ARs) and A2A receptors (A2ARs) are expressed only in adult ventricular myocytes. However, there is no definite evidence of the location of A3 or A2BARs on adult mammalian myocytes.[18] Adenosine exerts its cardioprotective action by binding to the A1 and A3 receptor located on the myocardial plasma membrane. Adenosine A1 and A3 receptors mediate inhibition of adenylate cyclase via a guanine nucleotide binding inhibitory protein (Gi/o). However, A2 receptor couple to a comparable stimulatory protein Gs. [19] Agonism of A2AARs on CD4+ T lymphocytes reduces the accumulation of neutrophils in ischemic tissues, this may leads to a reduction in the infarct size. Moreover, A3ARs have been identified in human eosinophils. Although one way of affording cardioprotection is the inhibition of resident mast cell degranulation, however, it is unclear whether A2BARs have a role in this process.[11] Moreover, A1 receptors coupled to G protein, to Phospholipase C (PLC)/ Protein kinase C (PKC) and further activation of PKC results the protection against ischemia reperfusion induced myocardial injury.[20]
Role of bradykinin
The initial release of bradykinin as protective mediators in the early stage of ischemia may also tribute to the protective effect of ischemic preconditioning. Ischemia leads to early generation of bradykinin, either from coronary vascular endothelial cells or through activation of plasma kallikrein, and this leads, via the activation of endothelial vascular bradykinin B2 receptors, to the generation of both prostacyclin (PGI2) and nitric oxide (NO). NO further has been diffuses to cardiac myocyte, stimulate soluble guanylyl cyclase with a resultant increase in cyclic guanosine monophosphate level leading to reduction in myocardial oxygen demand.[9] Bradykinin release is protective under condition of ischemia, is presumably enhanced during therapy with angiotensin- converting enzyme (ACE) inhibitors and is suppressed under condition of endothelial dysfunction.[21]
Bradykinin binding to GiPCR receptor may leads to the activation of P13-k, phosphorylation of Akt and ERK1/2, generation of nitric oxide, activation of PKG and opening of mitochondrial KATP channels. This may leads to the generation of reactive oxygen species (ROS) resulting in preconditioning. [9] The ACE inhibitor potentiate the beneficial cardiovascular and metabolic effect of endogenous kinins. The cardioprotective action induced by kinin or ACE inhibitor can be abolished with selective B2 kinin receptor antagonists.[22] Moreover bradykinin was reported to prevent reperfusion injury by activating P13 kinase. The inhibition of mitochondrial permeability transition pore (mPTP) opening may play a role in bradykinin induced cardioprotection during reperfusion.[17]
Role of opioids
The endogenous opioid peptide also provides cardioprotective effect of ischemic preconditioning. These receptors involved in regulating the cardiovascular system have been localized centrally to the cardiovascular and respiratory centres of the hypothalamus. Three types of opioid receptor (µ-, δ-, and k) have been characterized through biochemical and pharmacological methods. Opioid receptor are G-protein coupled receptor and activation of these receptor further inhibits the adenyl cyclase. The µ- opioid receptor activation may lead to the protection via a reduction of the inflammatory response including neutrophil activation, however δ-, and k-opioid activation leads to a granulocyte complex to induced preconditioning.[12]
Role of nitric oxide
Nitric oxide (NO) plays an important role in preconditioning-induced cardioprotection. The brief episodes of ischemia/ reperfusion have been reported to produce increased level of NO via nitric oxide synthase (NOS) in consequent activation of protein kinase C ε, tyrosine kinase may increase the production of nuclear factor kB (NF-κB) via cyclic guanosine monophosphate (cGMP)-dependent signalling pathways. NO have also been shown to attenuate ischemic reperfusion injury by regulating myocardial cellular levels by cyclic guanosine monophosphate (cGMP) dependent and independent signalling. [11] Moreover, NO mediates the effect of tissue kallikrein in improving cardiac function and limiting post infarction remodelling through inhibition of inflammation.[12]
Role of acetylcholine
Acetylcholine (ACh) as a potent pharmacological agent mimics ischemic preconditioning (IP) enabling hearts to resist infarction during a subsequent period of ischemia. ACh stimulate Gi/o-linked receptors and ultimately mediate protection by opening mitochondrial ATP-sensitive potassium channels with the generation of reactive oxygen species that subsequently activate protein kinase C (PKC).[23]
Role of Calcitonin gene-related peptide (CGRP)
Calcitonin gene-related peptide (CGRP), a major transmitter of capsaicin-sensitive sensory nerves, play an important role in the preconditioning induced by brief ischemia or hyperthermia or by some drugs, and α-CGRP seems to play a major role in the mediation of delayed preconditioning. The cardioprotection afforded by CGRP-mediated preconditioning is due to inhibition of cardiac tumor necrosis factor-α (TNF-α) production .[24]
Description of plant:
Spondias pinnata belonging to family Anacardiaceae (cashew family) commonly known as Wild mango. It is widely spread in the topical region. The administration of plant extract of Spondias pinnata (with dose 200mg/kg ) has been reported to have antibacterial and ulcerprotective [25] Additionally, the antioxidant activity of this plant has been reported by using 70% of methanolic extract of stem bark of Spondias mangnifera.[26]
Rationale
Ischemic heart disease will be the global burden on health care.
Spondias pinnata has already reported as an antioxidant which may be play an important role in cardioprotection.
Aim and objectives
To study the effect of ethanolic extract of stem bark of Spondias pinnata on ischemic preconditioning of heart.
Objectives:
To investigate the role of Spondias pinnata in cardioprotective effect of ischemic preconditioning.
To explore the potential of plant extract of Spondias pinnata
Material and method:
Isolated perfused rat heart:
Heparinise the rats 20 min before sacrificing the animal. Anesthetize the rats with thiopental sodium (40mg/kg/i.p) remove the heart and mount on a langendorff's apparatus. Isolated heart is then retrogradely perfused at constant pressure of 80 mm Hg with Krebs- Henseleit buffer pH 7.4, maintained at 370C, bubble with 90% O2. Maintain the flow rate at 7-9 mL/ min using Hoffman's screw. Enclose the heart in a double walled jacket and maintain the temperature of circulating water at 370C. Global ischemia is produced for 30 min by blocking the inflow of Krebs - Henseleit solution which is followed by reperfusion for 120 min. Collect the coronary effluent immediately, 5min and 30 min after reperfusion for estimation of lactate dehydrogenase (LDH) and creatine kinase (CK).
Estimation of infarct size
Remove the heart from Langendorff's apparatus. Both the auricle and root of aorta are excised and ventricle kept overnight at 00C. Slice the frozen ventricles into uniform section of 2-3 mm thickness. Incubate in 1% triphenyltertrazolium chloride (TTC) at 370C in tris buffer (pH 7.4) for 20min. TTC is converted to red formazone pigment by NADH and dehydrogenase enzyme and therefore the viable cells stained deep red. The infarct cells have lost the enzyme and cofactor and thus remain unstained or dull yellow. Place the ventricular slices between two glass plates. Place the transparent plastic grid with 100 squares in 1 cm2 above it. Calculate average area of the ventricular slice by counting the number of square on either side. Similarly count number of square falling over non stained dull yellow area. Infarct size is expressed as percentage of average ventricular area.[27]
Estimation of lactate dehydrogenase (LDH)
Principle
LDH catalyses the following reaction:
Lactate + NAD Pyruvate + NADH
The pyruvate so formed is coupled with 2, 4-dinitrophenylhydrazine (2, 4-DNPH) to give correspondence hydrazone which gave a brown colour in alkaline medium. The intensity of this colour was proportional to the amount of LDH activity and is measured spectrophotometrically at 440 nm.
Procedure
Standard Curve
Tube No.
1
2
3
4
5
6
7
Enzyme Activity (IU/L)
0
167
333
500
667
833
1000
Pyruvate solution (ml)
000
0.05
0.10
0.15
0.20
0.25
0.30
NADH solution (ml)
000
0.05
0.10
0.15
0.20
0.25
0.30
Buffered lactate (ml)
1.00
0.90
0.80
0.70
0.60
0.50
0.40
NAD solution (ml)
000
0.20
0.20
0.20
0.20
0.20
0.20
Distilled water (ml)
0.30
0.10
0.10
0.10
0.10
0.10
0.10
DNPH reagent (ml)
1.00
1.00
1.00
1.00
1.00
1.00
1.00
Mix and incubate at 37°C for 15 min
NaOH solution (ml)
10
10
10
10
10
10
10
All the tubes are vortexed and optical density is measured spectrophotometrically at 440 nm taking tube 1 as blank, standard curve is plotted taking enzyme activity on X-axis and optical density on Y-axis.
Control (C)
0.1 ml of distilled water is added to 0.5 ml of buffered lactate. The contents are mixed well and incubated at 37°C for 20 min. To the above solution, 0.5 ml of DNPH colour reagent and 0.05 ml of coronary effluent (collect immediately after reperfusion) are added. Contents are vortexed and incubated at 37°C for 15 min. Then 5 ml of sodium hydroxide solution is added and the mixture is allowed to stand at room temperature for 5 min.
Test (T)
0.05 ml of coronary effluent (collected after stabilization of heart, immediately or 30 min after reperfusion) is added to 0.05 ml of buffered lactate. Solution is incubated at 37°C for 5 min, after vortexing. To the above contents, 0.1 ml of NAD+ solution is added, vortexed and incubated at 37°C for 15 min. It is followed by addition of 0.5 ml of DNPH colour reagent and contents are incubated at 37°C for 15 min. Finally, 5 ml of sodium hydroxide solution is added. Contents are mixed thoroughly and allowed to stand at room temperature for 5 min.
Calculations
Absorbance of test (AT) and control (AC) is measured against distilled water at 440 nm.
Net absorbance of test (AN) = AT - AC
Enzyme activity is calculated from standard plot by marking AN on Y-axis and extrapolating it to corresponding enzyme activity on X-axis.
Estimation of creatine phosphokinase (CK)
Principle
CK catalyses the following reaction:
Creatine phosphate + ADP Creatine + ATP
At pH 7.4, CK catalyses the forward reaction. The creatine so formed, reacts with diacetyl and α-naphthol in alkaline medium to give pink coloured complex. The intensity of this colour is proportional to enzyme activity and is measured spectrophotometrically at 520 nm.
Procedure
Blank (B)
To 0.2 ml of magnesium acetate solution, 0.25 ml of creatine phosphate solution, 0.1 ml of cysteine, 0.1 ml of coronary effluent (collected after stabilization of heart and after 5 min of reperfusion) and 0.25 ml of distilled water are added. Solution is vortexed and incubated at 37°C for half an hour. To the above solution, 0.5 ml of p-chloromercuribenzoate, 2.5 ml of α-naphthol, 0.5 ml of diacetyl solution and 6 ml of distilled water is added. The solution is thoroughly mixed and incubated in dark (to avoid degradation of α-naphthol) at room temperature for 30 min.
Standard (S)
To 0.2 ml of magnesium acetate solution, 0.25 ml of creatine phosphate solution, 0.1 ml of cysteine, 0.1 ml of coronary effluent (collected after stabilization of heart and after 5 min of reperfusion) and 0.25 ml of working creatine solution are added. Solutions are vortexed thoroughly and incubated at 37°C for half an hour. To the above solution, 0.5 ml of p-chloromercuribenzoate, 2.5 ml of α-naphthol, 0.5 ml of diacetyl solution and 6 ml of distilled water are added. The solution is thoroughly mixed and incubated in dark (to avoid degradation of α-naphthol) at room temperature for 30 min.
Test (T)
0.2 ml of magnesium acetate solution, 0.25 ml of creatine phosphate solution, 0.1ml of cysteine, 0.1ml of coronary effluent (collected after stabilization of heart and after 5 min of reperfusion) and 0.25ml of ADP are added. Solution is vortexed and incubated at 37°C for half an hour. To the above solution, 0.5ml of p-chloromercuribenzoate, 2.5ml of α-naphthol, 0.5ml of diacetyl solution and 6ml of water are added. The solution is thoroughly mixed and incubated in dark (to avoid degradation of α-naphthol) at room temperature for 30min. After 30 min of incubation in dark, absorbance of test (AT), standard (AS) and blank (AB) are measured spectrophotometrically against distilled water at 520 nm.[28]
Calculations
AT- AB 103 x Creatine taken (μM)
CPK (IU/L) = x
AS - AB Incubation time x Vol. of coronary effluent
Grouping of animal: The animals are divided into six group are as follow:
Group 1: (Control group)
Group 2: (Ischemic preconditioning group)
Group 3: (Control + Dose d1 (100 mg/kg)
Group 4: (Control + Dose d2 (200 mg/kg)
Group 5: (Dose d1 (100 mg/kg) + Ischemic preconditioning)
Group 6: (Dose d2 (200 mg/kg) + Ischemic preconditioning)
Experimental part:
Soxhlet Extraction Procedure:
The plant was dried at room temperature and crushed into coarsely powdered form. Extraction of coarsely dried powder (100gm) was performed with 95% ethanol (350 ml) at 65-70°C for 3hrs and the extract obtained was air dried at room temperature.
Work plan
Literature review (done).
Collection, authentication of plant (done).
Extraction of plant (done).
Approval for animal studies form IAEC (done).
Activity studies (in process)
Data analysis (1month)
Compilation and thesis (2months)
