INTRODUCTION
Heart disease is one of the leading causes of mortality in the world (1). Studies have shown that oxidative stress, inflammation and cell death are the most important pathophysiological causes of diseases, such as heart failure and cardiomyopathy (2). It has been shown that increase in oxidative stress in turn raises the level of reactive oxygen species (ROS) in the heart tissue (1–3). Generation of energy in the mitochondria from electron transfer chain pathway is associated with H+ production, which increases the ROS levels, thereby increasing the pro-oxidant-antioxidant balance (PAB) protein and oxidative stress. This ultimately causes dysfunction in proton carriers, DNA damage and cell death (4–6).
The overexpression of O-6-methylguanine DNA methyltransferase (MGMT) that has a high affinity to free electrons, could indicate a decrease in O
2− levels (7,8). Studies have shown that under oxidative stress conditions, exercise reduces malondialdehyde (MDA) and improves antioxidant capacity and performance of electron transport chain proteins (8–10). In addition to exercise, the use of medicinal plants has been widely examined for this purpose mainly due to their limited side effects.
Portulaca oleracea from the
Oleracea family is a medicinal plant with favorable components including omega-3 fatty acids, alpha-linolenic acid, flavonoids, coumarins and betalin, which could protect cells against free radicals and lipid peroxidation. According to some studies, this plant also has anti-atherosclerotic effects (11,12). It is believed that the antioxidant-effects of this medicinal plant can reduce left ventricular blood pressure, MDA (13,14) and inflammatory factors (15). On the other hand, the simultaneous use of medicinal plants along with exercise activities have been suggested as a complementary therapy for treatment of heart disease (11).
Although the results of most studies on the effect of exercise on oxidative stress markers are contradictory (6,8,9,11), it has been well-established that combination therapy with exercise and healthy diet can improve oxidative stress. In the present study, we investigate effects of aerobic exercise and
P. oleracea (purslane) seed extract supplementation on mitochondrial function and oxidative stress markers in the heart tissue of H
2O
2-poisoned rats.
MATERIAL AND METHODS
In this clinical trial, 72 male Wistar rats (weighting 180 to 200 g) were purchased from Pasteur Institute of Iran. The animals were housed in the laboratory of the University of Tehran for one week to adapt to the environment. All animals were treated in accordance with the ethical codes of working with laboratory animals. The animals were kept in standard temperature (20 to 24 °C) and under 12-hour dark and 12-hour light cycles, with free access to food and water.
First, the rats were randomly divided into nine groups of eight including (1) control+H
2O
2 (
C+H2O2), (2) aerobic exercise (
EX+H2O2), (3) aerobic exercise and 50 mg/kg purslane seed extract (
EX+PS50+H2O2), (4) aerobic exercise and 200 mg/kg purslane seed extract (
EX+PS200+H2O2), (5) aerobic exercise and 400 mg/kg purslane seed extract (
EX+PS400+H2O2), (6) 50 mg/kg purslane seed extract (
PS50+H2O2), (7) 200 mg/kg purslane seed extract (
PS200+H2O2), (8)4 00 mg/kg purslane seed extract (
PS400+H2O2), and (9) control (
C) groups.
Rats in the experimental groups received peritoneal injection of 1mmol/kg H
2O
2 for eight weeks, three times a week (16). Groups 2 to 5 performed aerobic exercise three sessions a week for eight weeks, and rats receiving purslane seed extract were given intraperitoneal injections on a daily basis. The control group was considered to investigate the effects of H
2O
2 on the research variables in the heart tissue and received intraperitoneal normal saline injections.
Dry purslane seeds were obtained from the Research Institute of Medicinal Plants. The seeds were powdered with an electric mill and then steeped in 80% ethanol at 1:10 ratio in two one-hour steps. The mixture was then passed through a 0.2 mm paper filter. The remaining material was placed in the percolation device to evaporate ethanol (11) and finally diluted with normal saline for injection to rats.
The exercise groups 2-5 performed daily aerobic exercise on the treadmill for eight weeks. Rats were trained on the treadmill at speed of 8 m/min and 10° slope for 30 minutes in the first week, at 12 m/min with the same slope and duration in the second week, at 16 m/min with the same slope for 45 minutes in the third week, and at 20 m/min with the same slope for 45 minutes in the fourth week. From the fifth week until the end of the study period, the rats were trained at 20 m/min and 10° slope for 60 minutes (17).
Forty eight hours after the last exercise session and after 10-12 hours of fasting, biopsy was performed. First, the rats were anesthetized by intraperitoneal injection of ketamine (90 mg/kg) and xylazine (10 mg/kg). After cleaving the thoracic cavity, the heart tissue was carefully separated, washed with distilled water and weighed. Then, it was immediately placed at -70 °C until measurement of research variables. All sampling procedures began at 8:00 and completed at 11:30. It should be noted that all rats were sacrificed as quickly as possible with minimum pain.
The levels of ATP (Cat NO: KA1661; ABNOVA; Germany), MGMT (Cat NO: DL-MGMT-Ra; DEVELOP; China) and MDA (Cat NO: CSB-E08558r; CASOBIO; China) were measured using commercial ELISA kits. The Guimarães-Ferreira paper was used for measuring PAB (18).
Data were reported as mean and standard deviation. Normal distribution of data was assessed using the Shapiro-Wilk test. Independent sample t-test was used to determine the effect of H
2O
2 on the C and C+H
2O
2 groups. The main effect of exercise, purslane seed extract and their combination were assessed using two-way analysis of variance and the Bonferroni's post hoc test. All statistical analyses were performed in SPSS (version 19) and at significance level of 0.05.
RESULTS
The cardiac level of ATP (P=0.0001, t
18=32.760) and MGMT (P=0.0001, t
18=13.716 t) in the C group were significantly higher than in the C+H
2O
2 group. Also, the cardiac level of MDA (P=0.0001, t
18=54.833) and PAB (P=0.0001, t
18=32.834) in the C group was significantly lower than in the C+H
2O
2 group. Aerobic exercise (F
1,72=37.948, P=0.0001, ƞ=0.345) and purslane seed extract (F
1,72=147.610, P=0.0001, ƞ=0.860) each significantly increased cardiac ATP concentration in the H
2O
2-poisoned rats. Combination of aerobic exercise and purslane seed extract supplementation also significantly increased cardiac ATP level in the H
2O
2-poisoned rats (
F3.72=16.35, P= 0.0001, ƞ=0.405). According to the Bonferroni’s post hoc test, the effect of purslane seed extract on ATP level was dose dependent. Compared to the control group, supplementation with purslane seed extract significantly increased cardiac ATP levels at doses of 200 (P=0.0001) and 400 mg/kg (P=0.0001) but not at at 50 mg/kg (P=0.99). This effect was more profound at dose of 400 mg/kg compared to 200 mg/kg (P=0.0001) (Figure 1).
Aerobic exercise (F
1,72=77.957, P=0.0001, ƞ=0.520) and purslane seed extract (F
3,72=85.994, P=0.0001, ƞ=0.782) significantly increased cardiac MGMT concentrations in the H
2O
2-poisoned rats. Combination of aerobic exercise and purslane seed extract also significantly increased cardiac MGMT concentration in the H
2O
2-poisoned rats (F
3,72=21.143, P=0.0001, ƞ=0.468). Compared to the control group, purslane seed extract significantly increased cardiac MGMT concentrations at doses of 200 mg/kg (P=0.0001) and 400 mg/kg (P=0.0001) but not at 50 mg/kg (P=0.99). This effect was more profound at 400 mg/kg compared to 200 mg/kg purslane seed extract (P=0.0001) (Figure 2).
Aerobic exercise (F
1,72=606.307, P=0.0001, ƞ=0.894), purslane seed extract (F
3,72=750.344, P=0.0001, ƞ=0.969) and their combination (F
3,72=180.794, P=0.0001, ƞ=0.883) significantly reduced cardiac MDA concentration in the H
2O
2-poisoned rats. Compared to the control group, supplementation with purslane seed extract could significantly reduce (P=0.0001) MDA concentration at all tested doses with the highest effect observed at 400 mg/kg (Figure 3).
Aerobic exercise (F
1,72=665.628, P=0.0001, ƞ=0.902), purslane seed extract (F
3,72=407.634, P=0.0001, ƞ=0.944) and their combination (F
3,72=60.373, P=0.0001, ƞ=0.716) significantly reduced PAB concentration in the cardiac tissue of H
2O
2-poisoned rats. The effect of purslane seed on the reduction of cardiac PAB was dose dependent. Consumption of purslane seeds at 200 mg/kg (p=0.0001) and 400 mg/kg (p=0.0001) resulted in decreased cardiac PAB. Also, a dose of 400 mg/kg had a greater effect on cardiac PAB reduction than 200 mg/kg (Figure 4).