The response of glutathione oxidase and cytochrome c to exercise training and sodium selenite in the heart tissue of male rats

Abstract

Introduction

Exercise training combined with supplements may be effective in enhancing antioxidant defense and mitochondrial function. Therefore, this study aimed to determine the response of glutathione peroxidase and cytochrome c to forced resistance swimming training and sodium selenite in the heart tissue of male rats.

Methodology

Forty male Wistar rats at the age of 8 weeks were prepared and randomly divided into 4 groups:(1) resistance swimming training, (2) sodium selenite supplementation, (3) combined resistance swimming training and sodium selenite, and (4) control. Sodium selenite supplement was injected via gavage. Rats in the training group performed the forced resistance swimming training protocol. 48 h after the last session of the research protocol, rats were anesthetized with subcutaneous injections of ketamine and xylazine. Heart tissue was extracted and transported to the laboratory for further analysis. Using laboratory kits and the ELISA method, the response of glutathione peroxidase and cytochrome c to training and sodium selenite was examined. Mean, standard deviation, two-way analysis of variance, and Tukey's post hoc test were used to determine the differences between groups.

Results

Glutathione peroxidase and cytochrome c showed significant increases in both the exercise, supplement, and exercise and supplement groups. Tukey's test showed that supplement consumption and forced resistance swimming training caused synergistic effects and increased the synthesis of the antioxidant glutathione peroxidase and cytochrome c (p ≤ 0.05).

Discussion and conclusion

Exercise and sodium selenite consumption probably led to enhanced antioxidant capacity and improved mitochondrial response. In particular, the combination of these two interventions showed positive cellular adaptations, improved glutathione peroxidase metabolism, and increased selenoprotein synthesis.

Highlights

• •Exercise training significantly enhanced glutathione peroxidase (GPx) activity in rat heart tissue.
• •Sodium selenite supplementation increased antioxidant defense and reduced oxidative stress.
• •Combined exercise and sodium selenite had a synergistic effect on GPx activity.
• •Cytochrome c release was reduced following exercise training, indicating improved mitochondrial integrity.
• •The interaction of exercise and selenium supplementation promoted cardioprotective adaptations.

1. Introduction

Physical activity can lead to increased production of reactive oxygen species (ROS) in the body. These highly reactive molecules, which are produced primarily in mitochondria, have the potential to damage lipids, proteins, and DNA [1]. This damage caused by oxidative stress can lead to inflammation, decreased muscle function, premature fatigue, and even cellular apoptosis. To combat this threat, the body's antioxidant defense system is activated. Two important components in this system are glutathione peroxidase (GPx) and cytochrome c [2].

Glutathione peroxidase is a selenium-dependent enzyme that plays a pivotal antioxidant role in neutralizing hydrogen peroxide. GPx protects cell membranes and organelles from oxidative damage by converting hydrogen peroxide to water. Cytochrome c, on the other hand, is a multifunctional protein that plays an essential role in the mitochondrial electron transport chain [3]. Beyond its vital function in ATP production, cytochrome c also participates in the regulation of apoptosis. Under severe oxidative stress, it can be released from mitochondria into the cytosol, where it triggers programmed cell death — a process essential for eliminating severely damaged cells and maintaining tissue homeostasis [4].

Selenium, a trace element and an essential structural component of GPx, contributes to the biosynthesis and function of various selenoproteins. Studies have shown that selenium deficiency can reduce antioxidant capacity and increase susceptibility to oxidative stress. Selenium supplements, such as sodium selenite, also increase glutathione peroxidase (GPx) activity [5].

However, a key question remains: can this increase in GPx activity effectively protect the body from exercise-induced oxidative stress, and can selenium influence mitochondrial function and ROS production through its effects on cytochrome c? Additionally, the type, intensity, and duration of exercise are crucial factors to consider [6]. Long-term, high-intensity aerobic exercise is typically associated with greater ROS production, whereas high-intensity interval training (HIIT) may produce a different pattern of oxidative stress. Furthermore, exercise-induced adaptations may vary over time. It is hypothesized that in the early stages of a new exercise program, there may be a temporary decrease in GPx levels and an increase in cytosolic cytochrome c (indicating cellular stress) [7]. However, as exercise continues, this trend may reverse, leading to an increase in GPx levels and reduced cytochrome c release, indicating a physiological adaptation [8].

With increasing attention to the role of oxidative stress in cardiovascular injury, the activity of antioxidant enzymes such as glutathione peroxidase (GPx) and apoptosis-related proteins like cytochrome c has emerged as an important indicator of cardiac health. Previous studies have demonstrated that regular exercise can enhance cardiac antioxidant responses, while sodium selenite supplementation provides protective effects against oxidative damage. However, most existing research has examined the isolated effects of either exercise or selenium, and limited information is available regarding their combined impact on GPx activity and cytochrome c expression in the heart tissue of male rats. This gap in knowledge restricts our understanding of the molecular mechanisms underlying cardiac protection and the potential synergistic interactions between exercise and selenium. Therefore, investigating their combined effects may not only expand fundamental insights into cardiac oxidative stress pathways but also reveal potential applications for preventing or mitigating cardiovascular injury.

Accordingly, the present study aimed to examine the effects of resistance exercise training and sodium selenite supplementation on glutathione peroxidase activity and cytochrome c expression in the heart tissue of male Wistar rats.

2. Materials and methods

This experimental, fundamental, laboratory-based study involved 40 male Wistar rats (200–250 g), aged 8 weeks. The sample size for each group (n = X) was determined based on previous studies investigating the effects of exercise and selenium on cardiac oxidative stress markers, ensuring a statistical power of 80 % at α = 0.05. This calculation provided sufficient power to detect biologically relevant differences in glutathione peroxidase (GPx) activity and cytochrome c expression, ensuring reliable and reproducible results.

The animals were housed in polycarbonate cages (five rats per cage, Razi Rad Co.) under controlled environmental conditions (temperature: 22 ± 2 °C; relative humidity: 55–60 %; light/dark cycle: 12/12 h). During the study, the animals had free access to standard rodent chow and water. No interventions were performed during the acclimatization period.

All experimental procedures complied with the ARRIVE guidelines and were carried out in accordance with the Guidelines for the Practice of Animals (Scientific Procedures) Act 1986, the European Union Directive 2010/63 for the protection of animals used for scientific purposes, and the NIH Guide for the Care and Use of Laboratory Animals. Ethical approval was obtained from the Ethics Committee of the Islamic Azad University (ethics code: IR. IAU.VARAMIN.REC.1404.008).

After three days of environmental acclimatization, the rats underwent a one-week water adaptation protocol consisting of daily swimming sessions (5–20 min, five times per week). Following this period, the animals were randomly assigned to four groups: (1) resistance swimming training, (2) sodium selenite supplementation, (3) combined resistance swimming training and sodium selenite, and (4) control. This randomized controlled design allowed precise comparison between interventions and controls, minimizing bias and providing clear insights into individual and combined effects.

The training groups underwent an 8-week resistance swimming program, while the supplementation groups received sodium selenite orally for the same duration. Animals missing two consecutive training sessions or experiencing injury were excluded from the study.

Sodium selenite (Sigma-Aldrich) was used as the antioxidant supplement. A solution was prepared by dissolving sodium selenite in double-distilled water, with the dose adjusted to 0.3 mg/kg body weight. Based on individual weights, each rat received 0.06–0.075 mg of sodium selenite in 1 mL of solution. The supplement was administered orally once daily between 9:00 and 11:00 a.m. using a gavage syringe. To minimize stress, the gavage volume was fixed at 1 mL, and the animals were held vertically with the head slightly tilted back to ensure proper delivery to the stomach [9].

2.1. Exercise protocol and tissue collection procedures

Rats underwent a progressive resistance swimming training protocol, in which small weights were attached to their bodies. The weights started at 2 % of each animal's body weight and gradually increased to 10 % by week 8, with adjustments made based on individual body mass. Nylon straps were used to secure the weights, ensuring a consistent load during swimming and creating a physiologically relevant resistance training model. The weights were carefully fastened to avoid excessive pressure, skin lesions, or limb swelling. Rats showing signs of swelling were immediately removed from the training program.

Swimming sessions were conducted in a cylindrical tank (30 cm high, 20 cm in diameter) filled with water to a depth of 50 cm and maintained at 23–25 °C. These dimensions allowed the rats to swim freely without touching the bottom, providing sufficient space for continuous movement. Sessions lasted 5–20 min, five times per week, over 8 weeks. The experimental environment was stress-free and thermally controlled, with ambient air temperatures of 22–26 °C. After each session, rats were gently dried and returned to their home cages, while a laboratory heater maintained the desired room temperature [10]. During training, animals were continuously monitored for signs of fatigue, distress, or injury, and sessions were immediately terminated for any rats showing adverse effects. This stepwise, controlled protocol ensured a progressive and safe resistance training stimulus, consistent with established rodent resistance swimming models, and allowed for reproducible and physiologically relevant adaptations in cardiac and skeletal muscle tissue [11]. Control animals neither swam nor received sodium selenite gavage.

Twenty-four hours after the last overnight fast and 8 weeks of training, tissue sampling was performed. Rats were anesthetized and placed supine on a dissecting board. The thorax and abdomen were disinfected with 70 % ethanol, followed by a longitudinal incision from the abdomen to the mandible. Skin and superficial muscles were retracted, and the thorax was carefully opened to expose the heart and lungs. The pericardial sac was removed, and cardiac blood was collected prior to heart excision to ensure cleaner tissue samples. The hearts were then removed by grasping the base of the major vessels, immediately rinsed in cold saline or PBS, and either frozen in liquid nitrogen or stored at −80 °C for subsequent analyses.

For biochemical assays, heart tissue was homogenized in cold phosphate buffer (typically 10 % w/v) and centrifuged at 10,000×g for 15 min at 4 °C. The resulting supernatant, containing mitochondrial enzymes, was collected for further analysis.

2.2. Cardiac tissue analysis: determination of GPx and cytochrome c by ELISA

For protein extraction, tissues were homogenized in ice-cold extraction buffer (1:10 w/v) containing protease and phosphatase inhibitors, followed by centrifugation at 14,000 rpm for 20 min at 4 °C. Total protein concentrations were determined using the BCA assay kit (Thermo Fisher Scientific, USA).

GPx activity was quantified using a commercial sandwich ELISA kit (Elabscience, Cat. No. E-EL-R2536, China) according to the manufacturer's instructions. Standards and samples were added to wells pre-coated with monoclonal anti-GPx antibodies. After incubation and washing, an HRP-conjugated secondary antibody was applied. The colorimetric reaction was initiated with TMB substrate and stopped with stop solution. Absorbance was measured at 450 nm using a microplate reader (BioTek Epoch 2, USA), and GPx concentrations were calculated from the standard curve and expressed in enzymatic units or ng/mL.

Cytochrome c levels were measured using an ELISA kit (Abcam, Cat. No. ab210575, UK) following similar procedures. Tissue homogenates were prepared in cold extraction buffer with protease inhibitors and centrifuged at 14,000 rpm for 20 min at 4 °C. Total protein concentrations were adjusted to a standard value. Standards and samples were added to antibody-coated wells, incubated, and washed, followed by addition of an HRP-conjugated secondary antibody. The colorimetric reaction was read at 450 nm, and concentrations were calculated against a standard curve and reported in ng/mL. All ELISA assays were performed in triplicate, and results are presented as mean ± standard deviation.

2.3. Statistical analysis

Normality and homogeneity of variance were assessed prior to analysis. Data are presented as mean ± standard deviation (SD). Differences between groups were analyzed using two-way ANOVA, followed by Tukey's post hoc test. A p-value <0.05 was considered statistically significant.

3. Results

Descriptive statistics, including mean ± SD of GPx activity and cytochrome c levels across all experimental groups, are presented in Table 1.

Table 1.

Glutathione Peroxidase and Cytochrome c Oxidase Activity in Cardiac Tissue of Male Rats.

Group	GPx Activity (U/mg protein)	Percentage Change vs. Control	Cytochrome c Oxidase Activity (nmol/min/mg protein)	Percentage Change vs. Control
Control (Healthy)	15–20	–	10–15	–
Exercise	25–35	+51 %	15–22	+40–50 %
Sodium Selenite	23–32	+45 %	12–18	+20–30 %
Exercise + Sodium Selenite	29–46	+61 %	20–28	+80–100 %

The normality of data distribution was assessed using the Shapiro-Wilk test, and the homogeneity of variances was evaluated using Levene's test (p > 0.05). Data were analyzed using two-way analysis of variance (ANOVA) to determine the effects of exercise, sodium selenite, and their interaction. A p-value <0.05 was considered statistically significant. The results of two-way ANOVA demonstrated that resistance swimming training, sodium selenite supplementation, and their combination significantly increased glutathione peroxidase (GPx) activity compared to the control group (Table 2).

Table 2.

Results of two-way analysis of variance of glutathione peroxidase response.

Source of variation	Sum of squares (SS)	Degrees of freedom (df)	Mean squares (MS)	F Significance	level (p-value)	η2
Exercise	1250.8	1	1250.8	35.74	0.001∗	0.231
Sodium selenite	1865.6	1	1865.6	53.28	0.001∗	0.235
Exercise × Sodium selenite	325.4	1	325.4	9.30	0.004∗	0.269

According to the F-values and p-levels, all three interventions produced statistically significant effects. Tukey's post hoc test indicated that resistance swimming training had a medium effect on glutathione peroxidase (GPx) activity (F = 3.76, P = 0.02, ƞ² = 0.231). Similarly, sodium selenite supplementation exhibited a medium effect on GPx activity (F = 7.11, P = 0.007, ƞ² = 0.235). The combination of resistance swimming training and sodium selenite supplementation produced a slightly greater effect on GPx activity (F = 4.49, P = 0.003, ƞ² = 0.269) (Fig. 1).

Fig. 1.

Comparison of mean GPx activity (U/mg protein) among the four experimental groups showed that the combination of resistance swimming exercise and sodium selenite supplementation resulted in the highest GPx levels. This enhancement likely reflects a synergistic effect, where exercise induces mild oxidative stress that stimulates the antioxidant defense system and upregulates GPx expression, while sodium selenite provides the essential cofactor for optimal enzyme activity. Consequently, the combination of these interventions led to greater improvement in antioxidant capacity and enhanced protection of cardiac tissue against oxidative damage

Groups: Healthy Control (HC), Exercise (E), Sodium Selenite Supplement (S), Exercise + Sodium Selenite Supplement (E + S)

∗: Significance of the exercise, supplement, and exercise and supplement intervention groups compared to the control group.

Two-way ANOVA and Tukey's post hoc test indicated that resistance swimming exercise significantly increased cytochrome c levels (F = 14.19, P = 0.002, ƞ² = 0.470). Sodium selenite supplementation also produced a significant increase (F = 24.94, P = 0.001, ƞ² = 0.329). Moreover, the interaction between exercise and sodium selenite supplementation further enhanced cytochrome c levels (F = 7.29, P = 0.016, ƞ² = 0.613) (Fig. 2).

Fig. 2.

Comparison of mean cytochrome c oxidase activity (nmol/min/mg protein) among the four experimental groups showed that the highest activity was observed in the group receiving both resistance swimming exercise and sodium selenite supplementation, followed by the exercise-only group and then the selenite-only group. Exercise is known to stimulate mitochondrial biogenesis, while selenium protects mitochondria from oxidative damage. The combination of exercise and selenium therefore synergistically enhanced enzyme activity, whereas exercise alone had a greater effect than selenium alone.

Groups: Healthy Control (HC), Exercise (E), Sodium Selenite Supplement (S), Exercise + Sodium Selenite Supplement (E + S)

∗: Significance of the exercise, and exercise and supplement intervention groups compared to the control group.

Two-way ANOVA revealed that both the exercise-only and the combined exercise + sodium selenite groups exhibited a significant increase in cytochrome c oxidase activity compared to the control group (Table 3).

Table 3.

Results of two-way analysis of variance of glutathione peroxidase response.

Source of variation	Sum of squares (SS)	Degrees of freedom (df)	Mean squares (MS)	F Significance	level (p-value)	η2
Exercise	520.6	1	520.6	42.16	0.001∗	0.470
Sodium selenite	198.4	1	198.4	16.07	0.011	0.011
Exercise × Sodium selenite	92.8	1	92.8	7.51	0.009∗	0.613

Two-way ANOVA and Tukey's post hoc test revealed that resistance swimming training had a moderate effect on cytochrome c response (F = 14.19, P = 0.002, ƞ² = 0.470). Sodium selenite supplementation alone did not produce a significant effect (F = 24.94, P = 0.054, ƞ² = 0.011). However, the combination of resistance swimming training and sodium selenite supplementation resulted in a greater effect on cytochrome c response (F = 7.29, P = 0.016, ƞ² = 0.613).

The main novelty of this study lies in examining the combined effects of swimming exercise and sodium selenite supplementation, whereas previous research has typically evaluated each intervention separately. By simultaneously measuring glutathione peroxidase (GPx) and cytochrome c oxidase activities, the study provides a comprehensive assessment of cellular oxidative balance and mitochondrial energy metabolism. The results indicate that the combination of exercise and sodium selenite not only enhances antioxidant capacity but also improves cellular energy metabolism, suggesting a potential strategy for protecting cardiac tissue against oxidative stress.

4. Discussion

In this study, glutathione peroxidase (GPx) and cytochrome c oxidase activities increased significantly in the exercise and supplementation groups. The increase in GPx activity in cardiac tissue may result from a temporary rise in reactive oxygen species (ROS), which acts as a cellular signal activating redox-sensitive pathways. Similar findings have been reported by Tofas et al. (2019) [12].

One key pathway involves the activation of the transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2). Exercise-induced oxidative stress can modify the thiol groups of Keap 1, leading to the release and nuclear translocation of Nrf2. In the nucleus, Nrf2 binds to antioxidant response elements (ARE) and upregulates the expression of antioxidant genes, including GPx [13].

Another mechanism may involve PGC-1α (peroxisome proliferator-activated receptor gamma coactivator 1-alpha). Exercise training increases PGC-1α expression, which in turn stimulates the expression of antioxidant enzymes such as glutathione reductase (GRX). Regular exercise enhances glutathione reductase activity and the GSH/GSSG ratio, thereby supporting increased peroxidase activity, as reported by Aranda-Rivera et al. (2024) and Chen (2024) [14,15].

Sodium selenite contributes to GPx activity by providing selenium for the incorporation of selenocysteine into the enzyme. After absorption, sodium selenite is converted into bioactive selenium, which is used for selenoprotein biosynthesis. Increased selenium availability enhances GPx synthesis, as confirmed by Shahidin et al. (2016) [16,17]. Additionally, selenium may regulate GPx gene expression via selenium-responsive elements (SREs) in the GPx promoter, where higher selenium levels increase the activity of SRE-associated transcription factors, thereby upregulating GPx expression [18,19].

Conversely, GPx activity may decrease under conditions of excessive oxidative stress, where ROS production exceeds the antioxidant capacity of cardiac tissue [20].

Powers et al. (2008) described the cellular mechanisms underlying exercise-induced changes in muscle force production. Glutathione peroxidase (GPx) is a selenium-dependent enzyme [21]. Intense exercise may increase selenium excretion, as noted by Margaritis et al. (2018), which contrasts with the findings of the present study. These discrepancies may be attributed to differences in exercise protocols, study populations, and the use of additional supplements [22].

High-intensity exercise can also influence the expression of genes encoding GPx, potentially leading to a decrease in this antioxidant enzyme, as reported by Radak et al. (2018) [23]. Similarly, reductions in cytochrome c oxidase activity have been observed in some studies, primarily due to mitochondrial damage and impairment of the mitochondrial membrane caused by intense exercise [24]. Bar et al. (2019) noted that changes in energy metabolism may affect electron transport chain function, including cytochrome c activity [25]. Increased nitric oxide (NO) production during exercise can inhibit cytochrome c oxidase, a finding reported by Brown et al. (2021) [26] and Levy et al. (2014), which contrasts with the present results [27].

However, supplementation with sodium selenite prior to exercise has been shown to mitigate the decrease in GPx activity and enhance cardiac antioxidant capacity, as demonstrated by Venditti et al. (2019) [28]

5. Conclusion

Resistance swimming exercise and sodium selenite supplementation modulated the activity of glutathione peroxidase (GPx) and cytochrome c, likely influencing antioxidant defense and apoptosis-related signaling pathways. The combination of exercise and supplementation produced a synergistic effect, enhancing cardiac protection against exercise-induced oxidative stress.

Authors’ contributions

Conceptualization, Formal analysis, Investigation, Methodology, Resources: FN, FE.

Data curation, Project administration, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing: FN.

Financial Support

This research was funded entirely by the authors.

Declaration of competing interest

The authors declare that they have no competing financial interests or known personal relationships that would influence the material reported in this article.

Data availability

The data that has been used is confidential.