Document Type : Research Paper
Authors
1
Department of exercise physiology and corrective movements, Faculty of Sport Sciences, Urmia University, Urmia, Iran
2
Department of Basic Sciences, Faculty of Paramedical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran
3
Department of Nutrition, Faculty of Health, Qazvin University of Medical Sciences, Qazvin, Iran
Abstract
Background and Purpose
Type 2 diabetes mellitus (T2DM) is a common metabolic disorder characterized by insulin resistance, impaired insulin secretion, and chronic hyperglycemia that together promote tissue damage. Skeletal muscle, a major site of insulin-mediated glucose uptake, is especially vulnerable and in diabetic conditions shows reduced mass, impaired function, mitochondrial dysfunction, and activation of apoptotic pathways. Among the key regulators of apoptosis are the pro-apoptotic protein Bax and the anti-apoptotic protein Bcl-2, and their balance critically determines cell survival. High-intensity interval training (HIIT) is a time-efficient exercise modality that can improve insulin sensitivity, oxidative capacity, and cellular stress tolerance. Eryngium (Buganagh) is a medicinal plant rich in antioxidant and anti-inflammatory constituents that may help counteract oxidative stress and metabolic disturbances in diabetes. The present study investigated the independent and combined effects of HIIT and Eryngium extract on Bax, Bcl-2, and the Bax/Bcl-2 ratio in the soleus muscle of rats with type 2 diabetes.
Materials and Methods
This experimental study involved fifty male Wistar rats aged 8–10 weeks and weighing 200 ± 20 g. Animals were housed under standard laboratory conditions, including a temperature of 22 ± 2 °C, a 12-hour light/dark cycle, and humidity of 50–60%, with free access to chow and water. Before interventions, rats completed a two-week acclimatization period to the housing environment and treadmill running to reduce stress. Type 2 diabetes was induced by two weeks of high-fat diet followed by a single intraperitoneal injection of streptozotocin (50 mg/kg in citrate buffer, pH 4.5). Seventy-two hours later, fasting blood glucose was measured, and rats with values above 250 mg/dL were considered diabetic. Animals were randomly assigned to five groups (n = 10): healthy control, diabetic control, diabetic plus Eryngium extract, diabetic plus HIIT, and diabetic plus HIIT with Eryngium extract. The extract was administered orally by gavage at 100 mg/kg/day for six weeks. The HIIT protocol was performed on a motorized treadmill five days per week for six weeks using repeated high-speed bouts with active recovery, with intensity progressively increased. At the end, rats were fasted overnight, anesthetized with ketamine/xylazine, and euthanized according to ethical guidelines. Soleus muscles were excised, cleaned, snap-frozen, and stored at −80 °C. Bax and Bcl-2 protein levels were measured with commercial ELISA kits, and the Bax/Bcl-2 ratio was calculated as an apoptotic index. Body weight and glucose were monitored, treadmill intensity was adjusted individually, and investigators were blinded to group allocation during biochemical analyses. All procedures followed institutional animal care standards, ensuring humane treatment, minimized distress, and consistent handling throughout the study. Sample processing, data recording, and assay validation were performed under controlled laboratory conditions to maintain reliability, reduce variability, and ensure that measured apoptotic markers accurately reflected intervention effects on skeletal muscle tissue in this experiment.
Results
In this study, the soleus muscle was chosen because its oxidative and slow-twitch profile makes it particularly sensitive to diabetes-induced apoptotic alterations.The induction of diabetes produced marked alterations in apoptosis-related proteins within the soleus muscle. One-way ANOVA revealed that Bax protein levels were significantly higher in the diabetic control group compared to all other groups (p = 0.001). Specifically, streptozotocin (STZ) injection resulted in a profound elevation of Bax concentration in diabetic rats relative to healthy controls, confirming the pro-apoptotic impact of diabetes induction.Intervention analyses showed that both high-intensity interval training (HIIT) and the combined protocol of HIIT with Eryngium extract significantly reduced Bax levels in diabetic rats when compared to the diabetic control group (p = 0.001). Importantly, the reduction in Bax was more pronounced in the combined intervention group, demonstrating a significantly greater improvement than that observed in the extract-only group (p = 0.001). In contrast, administration of Eryngium extract alone did not produce a statistically significant reduction in Bax levels (p > 0.05), indicating that the extract by itself was insufficient to counteract diabetes-induced increases in pro-apoptotic signaling.Similarly, the one-way ANOVA for Bcl-2 revealed that healthy controls exhibited significantly higher Bcl-2 protein levels than diabetic controls (p = 0.001), reflecting the suppression of anti-apoptotic mechanisms under diabetic conditions. Treatment with Eryngium extract, HIIT, or their combination all resulted in significant increases in Bcl-2 levels compared to the diabetic control group (p = 0.001). However, no significant differences were observed among the three intervention groups (p > 0.05), suggesting that although all interventions enhanced anti-apoptotic signaling, none was superior in elevating Bcl-2 expression.Analysis of the Bax/Bcl-2 ratio further supported these findings. The ratio was markedly elevated in the diabetic control group relative to the healthy controls (p = 0.001), indicating a strong shift toward apoptosis. Both the HIIT group and the combined HIIT + extract group demonstrated significant reductions in the Bax/Bcl-2 ratio compared with diabetic controls (p = 0.001). Notably, the combined intervention resulted in a significantly greater reduction in the Bax/Bcl-2 ratio compared to the extract-only group (p = 0.001), highlighting the synergistic effect of combining exercise with Eryngium extract.Collectively, these results show that diabetes promotes a pro-apoptotic environment in skeletal muscle by increasing Bax and the Bax/Bcl-2 ratio while suppressing Bcl-2. HIIT—especially when combined with Eryngium extract—exerts stronger protective effects by attenuating pro-apoptotic markers and enhancing anti-apoptotic signaling more effectively than the extract alone.
Conclusion
This study demonstrated that the combination of high-intensity interval training and Eryngium extract effectively attenuated apoptosis in the soleus muscle of rats with type 2 diabetes. Diabetes induction markedly increased Bax, decreased Bcl-2, and elevated the Bax/Bcl-2 ratio, while both HIIT and the combined intervention significantly reversed these detrimental changes. The extract alone was less effective in lowering Bax but still enhanced Bcl-2, indicating that exercise provided the primary protective stimulus and the plant extract acted mainly as a complementary support. Overall, the combined protocol produced the most favorable profile of apoptotic markers, suggesting improved cellular survival under diabetic conditions. These findings highlight the potential of integrating structured high-intensity exercise with antioxidant herbal supplementation as a non-pharmacological strategy to limit diabetes-related muscle complications and may provide a rationale for future translational and clinical studies targeting diabetic sarcopenia and functional decline. In practice, such combined interventions may help preserve muscle function.
Article Message
The growing burden of type 2 diabetes demands practical, low-cost strategies to protect vulnerable tissues such as skeletal muscle. In this experimental model, combining high-intensity interval training with Eryngium extract reduced pro-apoptotic signaling, increased anti-apoptotic defenses, and improved the Bax/Bcl-2 balance in the soleus muscle of diabetic rats. These findings suggest that structured high-intensity exercise, particularly when paired with antioxidant herbal supplementation, may mitigate diabetes-related muscle damage and delay progression toward diabetic sarcopenia. Further clinical research is needed to determine optimal protocols and to clarify the translational relevance of this combined non-pharmacological approach. Future work should refine dose and intensity.
Ethical Considerations
All experimental procedures involving animals were carried out in accordance with the guidelines of the Laboratory Animal Ethics Committee of the University of Urmia. The study was approved under Ethics Code: IR-UU-AEC-3/53. All stages of work with laboratory animals were performed strictly according to the committee’s instructions to ensure humane care, minimization of pain and distress, and adherence to national and institutional standards for animal research. Further information about the ethical framework can be obtained from the Laboratory Animal Ethics Committee of the University of Urmia.
Authors’ Contributions
Conceptualization: Ghassan Amer Bedno, Asghar Tofighi
Data Collection: Ghassan Amer Bedno, Bahram Jamali, Mohammad Reza Shiri-Shahsavar
Data Analysis: Ghassan Amer Bedno, Bahram Jamali, Asghar Tofighi
Manuscript Writing: Ghassan Amer Bedno, Asghar Tofighi
Review and Editing: Asghar Tofighi, Mohammad Reza Shiri-Shahsavar
Responsible for Funding: Asghar Tofighi
Literature Review: Ghassan Amer Bedno
Project Manager: Asghar Tofighi
Any Other Contribution: All authors contributed to the interpretation of findings and approved the final version of the manuscript.
Conflict of Interest
The Authors Declare no Competing Financial or Professional Interests.
Acknowledgments
We extend our gratitude to all individuals who supported and facilitated this study.
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