The Effect of Caffeine Supplementation and High Intensity Interval Trainingon Myocardial Oxidation Stress in Male Wistar Rats

The enzymatic defense system includes the enzymes glutathione peroxidase, catalase, and superoxide dismutase, which are responsible for intracellular protection [1]. One of the most important products of lipid peroxidation is malondialdehyde, which has been of great interest and is considered the main indicator of oxidative stress [4, 5]. Aerobic and endurance exercises can improve tissue resistance against harmful stimuli that improve intracellular oxidative stress and apoptosis [7, 8]. Following intense physical activities, with an increase in reactive oxygen species and lipid peroxidation, including malondialdehyde, and an imbalance between oxidative stress and antioxidant defense, a decrease in total antioxidant capacity occurs [11]. Caffeine increases heart protection by negatively regulating inflammatory and apoptotic pathways in rats [13]. Songstad et al. showed that performing six weeks of intense interval training caused no significant difference in the amount of total antioxidant capacity and malondialdehyde in the liver and heart tissue of pregnant rats [12]. According to the conflicting results of the research regarding the effect of intense intermittent exercise on cardiac oxidative stress indicators and/or the use of caffeine supplements on oxidative stress indicators, it is necessary to conduct a study in order to find an effective non-pharmacological solution to reduce these indicators on the heart of rats. Therefore, the present research was done to investigate the effect of eight weeks of intense intermittent exercise and caffeine supplementation on the levels of malondialdehyde and the activity of superoxide dismutase and catalase enzymes in the heart tissue of male rats.

Methods :

In this experimental study, 40 male Wistar rats with an average age of 8 weeks and an average weight of 200-220 grams were purchased from the Center for Research and Breeding of Laboratory Animals at Pasteur Institute, Tehran. After two weeks of familiarization with the environment and how to perform the activity, the rats after weight matching were randomly divided into four groups (n=8) including 1- healthy control (C), 2- exercise, 3- caffeine supplement, and 4- caffeine supplement. The training program included running on a treadmill in two-minute intervals for eight weeks and every week for five days. The warm-up and cool-down phase at the beginning and end of the main phase of the exercise was performed with an intensity of 40-50% of the maximum speed (16-20 m/min) for 5 minutes on the treadmill. Animals performed high-intensity interval training (5 to 12 2-minute bursts with an intensity of 90-85% of the maximum running speed and a minute of rest between bursts and 10 meters per minute equivalent to 30-40% intensity of the maximum oxygen consumption) at 19-20 pm, on a treadmill [9]. The control groups did not participate in any activity program. Then, 48 hours after the last training session. a part of the left ventricular tissue of the heart was carefully removed and frozen in nitrogen at -80°C. Pure anhydrous caffeine powder (dry) prepared from Merck, German was administrated according to the body weight of the animals (70 mg per kilogram of body weight) by intraperitoneal injection from 15:00 to 17:00 for eight weeks and five days per week [14]. Each 100 mg of tissue was homogenized in 1 ml of saline phosphate buffer containing antiprotease cocktail by a homogenizer, and then the tissue was homogenized in Rcf 10,000 for 15 minutes at 4 °C and the supernatant was collected and used to measure the biochemical indices of the research. 

Statistical Analysis:

The Kolmogorov-Smirnov test was used to check the normal distribution of the data. To investigate the difference between groups, a one-way analysis of variance and Tukey's post hoc test were performed at a significance level of less than 0.05 using SPSS software.

The results of the one-way analysis of variance showed a significant difference in the levels of malondialdehyde (P<0.001 and F=508.808) and the activity of superoxide dismutase (P<0.001 and F=115.266) and catalase (P<0.05) between groups. The results of Tukey's test showed that the level of malondialdehyde in the caffeine group (P=0.081) did not change significantly compared to the control group, but it decreased significantly in the training group (P=0.01). Following high-intensity interval training and caffeine consumption, the level of malondialdehyde decreased significantly (P=0.0078) compared to the control group, although the decrease in malondialdehyde levels in the caffeine+training group was not significant compared to the caffeine and training groups (P=0.07). The levels of superoxide dismutase in the caffeine (P=0.02), training (P=0.012), and training+caffeine (P=0.0083) groups had a significant increase compared to the control group and the simultaneous effect of exercise and caffeine was not significant compared to the caffeine and training groups (P=0.14). Also, catalase levels increased significantly in the research groups compared to the control group (P=0.019 in the caffeine group, P=0.001 in the training group, and P=0.0094 in the training+caffeine group).

Discussion :

In this study, there was a significant decrease in the levels of the oxidative index, malondialdehyde, and a significant increase in the activity of the antioxidant enzymes superoxide dismutase and catalase in the heart tissue of the caffeine+training group in comparison with the healthy control group. In the present study, an increase in the levels of superoxide dismutase was observed following caffeine consumption, which was associated with a decrease in the levels of malondialdehyde. This result is inconsistent with the findings of Ashrafi et al. [16] and Saker et al. [15], possibly due to the amount of caffeine consumed. In line with the results of this research, Bafghi et al. showed that eight weeks of interval training in 3-minute intervals with an intensity of 80% oxygen consumption and for 20 minutes in each session, along with curcumin supplementation, significantly increased the activity levels of superoxide dismutase and catalase enzymes [22].