Plant collection and identification
The roots of Potentilla reptans L. were collected from Tangrah village located in the North region of Iran. A voucher specimen (45815-TUH) was deposited in Tehran University, Iran [6,7].
Preparation of plant extract
The extraction process of Potentilla reptans root was performed as previously described [7]. The P. reptans ethyl acetate fraction was dissolved and sonicated in water.
Animal care
Male Wistar rats (220–260 g, 7-8 weeks old) were obtained from the animal center, Golestan University of Medicinal Sciences, Gorgan, Iran. Rats were maintained in cage (3-4 rats/cage) under standard conditions with a 12-h light-dark cycle, an ambient temperature of 20–23 °C, and 40–50% humidity. The animals had free access to normal food and water. Animal procedures were performed according to the guidelines of the National Institutes of Health (NIH Publication No. 85-23, revised 2011) and ARRIVE guidelines [11-13]. All experimental protocol was approved by the Animal Ethics Committee (IR.GOUMS.REC.1394.149 and No.: 94.61.138) of Golestan University of Medical Sciences, Golestan, Iran [6,7].
Drugs
Evans blue, NaCl, NaHCO3, KCl, NaH2PO4, CaCl2, MgCl2, Glucose, and 2, 3, 5-triphenyl-tetrazolium chloride (TTC) were all purchased from Merck (Kenilworth, NJ). L-NAME (Nω-nitro-L-Arginine methyl ester-a nonspecific NOS inhibitor) was bought from Sigma-Aldrich (Billerica, MA). Wortmannin (Wort; the PI3k/Akt inhibitor), PD98059 (PD; the ERK1/2 inhibitor), Tyrphostin AG490 (the JAK/STAT3 inhibitor), and 5-hydroxy decanoate (a mitoKATP channel blocker) were purchased from “Santa Cruz Biotech, Dallas, Texas, USA”. The antioxidant determination Kits were purchased from ZellBio GmbH (Germany). The HPLC-grade solvents for column chromatography (CH3CN, MeOH, and acetone), were purchased from Sigma-Aldrich, Germany. All compounds were water-soluble.
Isolated hearts
All surgical procedures used in the present study had been previously explained [2,4,6,7]. Briefly, animals were anesthetized with a combination of 10% ketamine and 1% xylazine (100/10 mg/kg, IP) and then, heparin (200 IU/kg, IP) was injected as an anti-coagulation agent. After 1 min, surgery was performed when rats were completely anesthetized and did not show any toe pinch and corneal reflex. The exposed trachea was connected to a rodent ventilator (Model 683, Harvard Apparatus, Holliston, MA, USA) by a fit cannula. The aorta was cannulated and the hearts were rapidly excised and immersed in ice-cold Krebs-Henseleit buffer. Then, the hearts were implanted on Langendorff perfusion system at a constant pressure of 75 - 80 mmHg (depending the coronary flow) with an oxygenated (95% O2, 5% CO2, 37 ℃) Krebs-Henseleit buffer (in mM: 118 NaCl, 25 NaHCO3, 4 KCl, 1.2 NaH2PO4, 1.9 CaCl2, 1.2 MgCl2, 11.1 Glucose, pH 7.35–7.45). The hearts were perfused with the Krebs-Henseleit buffer for 30 min. Next, a 30 min regional ischemia was performed in the left anterior descending (LAD) artery by occlusion using a silk string (6–0 mm), followed by 100 minutes (mins) of reperfusion.
Experimental protocol
Rats were randomly divided into 13 groups. The sample size was calculated 6 sample per group using G*Power software (α = 5%, effect size =0.63, power = 90%). The hearts were perfused and stabilized (baseline), then the hearts were subjected to 30 mins of global ischemia induced by the left anterior descending (LAD) artery occlusion using a silk string (6-0 mm), followed by 100 mins reperfusion. The following protocols were performed: 1: IR (rats that received no treatment); 2: ischemic postconditioning (IPOST), which was achieved by 3 episodes of 10 seconds ischemia and 10 seconds reperfusion at the onset of the reperfusion phase; 3: Etpost (2µg/ml) was added in Krebs-Henseleit buffer and applied through the first 15 mins of the reperfusion phase; 4: Etpost (2µg/ml)+L-NAME(50µM); 5: Etpost(2µg/ml)+Wort(400 nM); 6: Etpost(2µg/ml)+PD(400 nM); 7: Etpost(2µg/ml)+AG (tyrphostin AG490, 400 nM); 8: Etpost (2µg/ml)+ 5HD (1μΜ); 9: L-NAME(50µM)+ IPOST +IR; 10: Wort(400 nM)+IPOST+IR; 11: PD(400 nM)+IPOST+IR; 12: AG490(400 nM)+IPOST+IR; 13: 5HD(1μM)+IPOST+IR. The inhibitors were applied individually, at 10 mins prior to the induction of global ischemia and in combination with Etpost during the first 15 mins of the reperfusion phase [2,4] as shown in Fig. 1. Based on our previous study the effective concentration (EC50) of ethyl acetate fraction of P. reptans (Et) was determined at 2µg/ml in a preconditioning mechanism. Thus, we used this Etpost at a concentration of 2µg/ml [7].
Hemodynamic parameters
A latex water-filled balloon was used to measure hemodynamic parameters, inserted into the left ventricle which was connected to a pressure transducer. The heart rate (HR), the left ventricular developed pressure (LVDP = LVSP − LVEDP), the left ventricular systolic pressure (LVSP), the rates of pressure development and fall (dP/dt max and dP/dt min), and the rate pressure product (RPP = LVDP × HR ÷ 1000) were monitored by the Power Lab software (Power Lab 8/30 AD Instruments, Australia) [2]. The coronary flow (CF) was measured at the end of the reperfusion period.
Infarct size determination
To identify infarct size and area at risk, at the end of the reperfusion period, TTC staining (2, 3, 5-triphenyl-tetrazolium chloride) was performed as previously described [7]. The infarct size was determined using the computerized planimetry technique (Photoshop, ver. 7.0, Adobe system, San Jose, CA, USA). The infarct size (IS) was measured and is expressed as a percentage of the area at risk (IS/AAR%), while the total area at risk (AAR) was expressed as the percentage of the ratio of the left ventricle (AAR/LV%) [2,7].
Real time PCR analysis for Nrf2
The expression of Nrf2 genes was investigated on the left ventricular myocardial tissue on the 7300 ABI Real-Time PCR system (Applied Biosystems, Foster City, California, USA). The primer sequence used in this study was the same as the primers applied in our previous research and are listed in Table 1. The target gene expression was normalized against the housekeeping gene GAPDH, and the relative expression was determined using the -2 Delta Delta Ct (DDCt) method [7].
Assessment of oxidative stress
The frozen tissue was lysed and homogenized in lysis buffer and then centrifuged to precipitate the insoluble materials. The resulting supernatant was employed for the assay. The concentration of malondialdehyde (MDA), as well as the endogenous antioxidants, namely SOD and CAT, was assessed by the ELISA method using commercial kits (ZellBio GmbH, Germany) according to their manufacturer's recommendations [7].
Assessment of ventricular arrhythmias
Ventricular arrhythmias were evaluated in accordance with the Lambeth Conventions [7]. Three forms of ventricular arrhythmias were analyzed; the premature ventricular complex (PVC) and bigeminy, ventricular tachycardia (VT) and ventricular fibrillation (VF), as well as arrhythmia score [2,4].
Immunohistochemistry
The immunohistochemical analysis was performed, as previously described for the assessment of BAX, BCl-2, and Caspase-3 expression in cardiomyocytes resident in the LV [7].
TUNEL staining
Total caspase 3 abundance in the rat heart sections was measured using the TUNEL assay (In-Situ Cell Death Detection Kit) according to the protocols introduced by previous studies [4,7].
Western blot analysis
Western blot analysis was performed on the LV sections to evaluate the expression of BAX (LS-C353915, LSBio, Seattle, US), BCl-2 (BS-4563R, Bioss Inc, Boston, US), Caspase3 (SC-56046, Santa Cruz Biotechnology, CA, US), GSK-3β (NBP1-47470, Novus Biologicals, Manama, US) and SGK1 (SC-28338, Santa Cruz Biotechnology, CA, US) proteins, as previously described in detail elsewhere [7]. GAPDH (ab181602, Abcam, Cambridge, US) was used as the loading control. The intensity of each protein band was semi-quantified by the Image J image analysis system.
Statistical assessment
Data were expressed as means ± SEM and analyzed by Graph Pad-Prism 6 software (San Diego, CA) and SPSS 16. The differences between groups were evaluated by using a one-way analysis of variance and the post hoc Tukey test. Hemodynamic data within and between groups were performed with a two-way analysis of variance. Kruskal-Wallis and Fisher’s exact test was applied for analyzing arrhythmia scores and VF incidence, respectively. P values < 0.05 were the considered significant levels.